in-ovo-intravascular-injection-in-chicken-embryos

<em>In Ovo</em> 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 ...

yangzhou university

Yangzhou University

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.

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

Spermatogonial stem cells (SSCs) are the basis of spermatogenesis. Systematically exploring the critical factors associated with the formation of SSCs will provide new insight to improve the formation efficiency, and their practical application. Here we explore the regulatory mechanism of the ECM-receptor interaction signaling pathway and related genes during differentiation of SSCs in chicken. Firstly, the positive cell rate of SSCs protein marker was detected by immunofluorescence and flow cytometry and qRT-PCR was used to identify, the expression of related marker genes after 10 days of RA-induction. Secondly, the ESCs on 0d/ 4d /10d after RA- induction/self-differentiation were collected, and the total RNA was then extracted from cells. Finally, high-throughput analysis methods (RNA-seq) were used to sequence the transcriptome of these cells. After PCA analysis of the RNA-seq data, Venny analysis, GO and KEGG enrichment were further used to find the key signaling pathways and genes in the RA-induction process. The results showed that on day 10 of RA-induction, grape cluster growth cells expressed integrinβ1, the specific marker protein of SSCs cells, and the integrinβ1 positive rate was 35.1%. Also, SSCs marker genes CVH, Integrinβ1, Integrinα6 were significantly up-regulated during RA-induction. Moreover, the significantly enriched pathway, ECM-receptor interaction signaling, in current study may play a crucial role in RA-induction. Then, JASPAR was used to predict the differential gene transcription factors in the signaling pathway, finding that RA receptor was a transcription factor of COL5A1, COL5A2 and COL3A1. The qRT-PCR results showed that the expression levels of RA receptors (RXRA, RARA and RXRG) and the predicted genes (COL5A1, COL5A2 and COL3A1) were both significantly increased during RA-induction. Also, dual-luciferase reporter assay showed that RA could affect the luciferin activities of COL5A1, COL5A2 and COL3A1. These results suggest that RA plays a crucial role in the formation of chicken spermatogonial stem cells via the transcription levels of COL5A1, COL5A2 and COL3A1 to regulate the ECM-receptor interaction signaling pathway. Additionally, knockdown of COL5A1/COL5A2/COL3A1 could effectively reduce the formation efficiency of SSCs. This indicated that the interference of RA receptor binding genes in the ECM-receptor interaction signaling pathway could decrease the efficiency of RA induced SSCs formation. Therefore, this study concludes that RA promotes formation of chicken spermatogonial stem cells by regulating the ECM-receptor interaction signaling pathway.

Qishenng Zuo

yangzhou university

In Ovo Intravascular Injection in Chicken Embryos