DNA hypomethylation activation Wnt/TCF7L2/TDRD1 pathway promotes spermatogonial stem cell formation.

Detailed analysis of the regulatory mechanism of spermatogonia stem cell (SSCs) genesis can provide a novel strategy for the application of SSCs in the fields of transgenic animal production and regenerative medicine. Previous studies in this study showed that WNT signaling can positively regulate the formation of SSCs, but the exact regulatory mechanism is not clear. Here, we predicted the target gene of the Wnt/TCF7L2 pathway, namely TDRD1, by bioinformatics analysis. Functional studies revealed that overexpression of TDRD1 during RA-induced SSCs formation in vitro significantly upregulated the expression of reproductive marker genes (Integrinß1 and Integrinα6), and further flow cytometric analysis also confirmed that the formation efficiency of SSCs was significantly increased after overexpression of TDRD1; while interference with TDRD1 showed the exact opposite result. The in vivo experiments were consistent with the results of the in vitro experiments. Interestingly, although Wnt/TCF7L2 can promote the formation of SSCs, its function must be dependent on the expression of TDRD1, which was also repeatedly demonstrated as a target gene of the Wnt/TCF7L2 signaling pathway. Mechanistically, we found a large number of CpG sites in the TDRD1 promoter, and BSP analysis also confirmed that DNA methylation modifications in the TDRD1 promoter were significantly higher in embryonic stem cells than in SSCs, and further dual-luciferase reporter system assays revealed that low DNA methylation modification levels could enhance TDRD1 promoter activity; although previous studies demonstrated that TCF7L2 could enrich in the TDRD1 promoter region, the binding of the two was dependent on low DNA methylation modification. Taken together, we confirmed that low DNA methylation mediates Wnt/TCF7L2 regulation of TDRD1 to promote the formation of SSCs, providing a basis for SSCs in improving animal productivity.

In Ovo Intravascular Injection in Chicken Embryos.

As a classical model system of embryo biology, the chicken embryo has been used to investigate embryonic development and differentiation. Delivering exogenous materials into chicken embryos has a great advantage for studying gene function, transgenic breeding, and chimera preparation during embryonic development. Here we show the method of in ovo intravascular injection whereby exogenous materials such as plasmid vectors or modified primordial germ cells (PGCs) can be transferred into donor chicken embryos at early developmental stages. The results show that the intravascular injection through the dorsal aorta and head allows injected materials to diffuse into the whole embryo through the blood circulatory system. In the presented protocol, the efficacy of exogenous plasmid and lentiviral vector introduction, and the colonization of injected exogenous PGCs in the recipient gonad, were determined by observing fluorescence in the embryos. This article describes detailed procedures of this method, thereby providing an excellent approach to studying gene function, embryo and developmental biology, and gonad-chimeric chicken production. In conclusion, this article will allow researchers to perform in ovo intravascular injection of exogenous materials into chicken embryos with great success and reproducibility.

Tle4z1 Facilitate the Male Sexual Differentiation of Chicken Embryos.

Background: Sex differentiation is a complex and precisely regulated process by multiple genes in chicken. However, it is still unclear on the key genes of sex differentiation. Objective: To explore the function of Tle4z1 screened by RNA-seq sequencing on sex differentiation during the development of chicken embryos. Methods: Tle4z1 was differentially expressed from the RNA-seq of ESCs and PGCs in male and female chickens. Then, we established an effective method to overexpression or knocking down the expression of Tle4z1 in ovo and in vitro, respectively. Histomorphological observation, qRT-PCR and ELISA were applied to detect the function of Tle4z1 in the process of male sex differentiation by injecting vectors into embryos at day 0. Results: It showed that Tle4z1 has significant male preference in embryonic day 4.5, such phenomenon persisted during the growth period of chicken embryos. Morphological observation results showed that the gonads on both sides of genetic male (ZZ) embryos with Tle4z1 knocking down developed asymmetrically, the gonadal cortex became thicker showing the typical characteristics of genetic female (ZW) gonads. Furthermore, the expression of Cyp19a1, which dominates female differentiation, was significantly increased, while the expression of male marker genes Dmrt1, Sox9, WT1 and AR was significantly downregulated. In addition, the concentration of testosterone also significantly decreased, which was positively correlated with the expression of Tle4z1 (P < 0.01). Conversely, the ZW embryo showed defeminized development when Tle4z1 was overexpressed. Conclusion: We prove that the Tle4z1 is a novel gene through the male sexual differentiation via gene regulation process and synthesis of testosterone, which construct the basis for understanding the molecular mechanism of sex differentiation in chickens.

Epigenetic modification cooperates with Zeb1 transcription factor to regulate Bmp4 to promote chicken PGCs formation.

BMP4 is the critical gene of primordial germ cell formation in mammal, however, the mechanism of PGCs formation in chicken still unknown. In this research, we compared the evolution relationship of different species. Although the protein sequence is highly conservative between mouse, human and chicken, promotors vary among avian and mammal species. Therefore, it is easily to predict that there would be different regulation mechanism of Bmp4 expression in chicken. Here, we elucidate the function of chicken Bmp4 during PGCs formation. In vivo, Bmp4 can promote PGCs development and migration, and increase the expression of key genes (Cvh, c-kit, cxcr4, etc.). Whereas, the expression of these genes will decrease after knocking out Bmp4. After over-expression and knockout Bmp4 in vitro, we found that overexpression of Bmp4 could promote the formation of embryoid bodies (EB) and up-regulate the key genes of PGCs formation and migration, while knockout Bmp4 could inhibit the formation of embryoid bodies and decrease the expression of related genes. Flow and indirect immunofluorescence also indicated the same result. These all results proved that chicken Bmp4 could also promote the formation of PGCs. Furthermore, dual-luciferase activity detection showed that the promotor activity of Bmp4 was positively regulated by transcription factor Zeb1. Overexpression of Zeb1 can also increase the mRNA and protein expression of Bmp4. At the same time, DNA methylation inhibited Bmp4 transcription and histone methylation was able to promote its transcription. In conclusion, this study established that chicken Bmp4 can promote the formation of chicken PGCs. This gene is regulated by DNA, histone methylation and transcription factor Zeb1. These results lay a theoretical foundation for exploring the function and molecular mechanism of Bmp4 in the process of PGCs formation.

H3K4me2 Promotes the Activation of lncCPSET1 by Jun in the Chicken PGC Formation.

Primordial germ cells are the ancestors of female and male cells. Current research has shown that long non-coding RNA (lncRNA) and Histone methylation are the pivotal epigenetic factors in the PGC formation. However, there are few studies on the regulatory mechanism of lncRNA in the formation of PGC. Here, we define the lncRNA highly expressed in chicken PGC, lncCPSET1 (chicken-PGC-specifically-expressed transcript 1) This study found that compared with the interference of lncCPSET1/histone methylase Mll2 alone, the PGC formation was severely inhibited with the interference of lncCPSET1 and histone methylase Mll2 jointly in vivo and in vitro. Studies on the transcription level of lncCPSET1 found that H3K4me2 and transcription factor Jun have a positive effect on the activation of lncCPSET1; while DNA hypomethylation inhibits the expression of lncCPSET1. In terms of mechanism, compared with DNA methylation, H3K4me2 dominates lncCPSET1 activation. H3K4me2 can be enriched in the lncCPSET1 promoter, change its chromosome conformation, recruit the transcription factor Jun, and activate the expression of lncCPSET1. Taken together, we confirmed the model that H3K4me2 rather than DNA hypomethylation mediates Jun to regulate lncCPSET1 transcription, which broadens the study of lncCPSET1 pre-transcriptional mechanism.