Abstract
Spermatogonial stem cells (SSCs) may apply to gene therapy, regenerative medicine in place of embryonic stem cells (ESCs). However, the application of SSCs was severely limited by the low induction efficiency and the lack of thorough analysis of the regulatory mechanisms of SSCs formation. Current evidences have demonstrated multiple marker genes of germ cells, while genes that specifically regulate the formation of SSCs have not been explored. In our study, cadherin-like and PC-esterase domain containing 1 (Cped1) expressed specifically in SSCs based on RNA-seq data analysis. To study the function of Cped1 in the formation of SSCs, we successfully established a CRISPR/Cas9 knockout system. The gene disruption frequency is 37% in DF1 and 25% in ESCs without off-target effects. Knockout of Cped1 could significantly inhibit the formation of SSCs in vivo and in vitro. The fragment of −1050 to −1 bp had the activity as Cped1 gene promoter. Histone acetylation could regulate the expression of Cped1. We added 5-azaeytidi (DNA methylation inhibitors) and TSA (histone deacetylase inhibitors) respectively during the cultivation of SSCs. TSA was validated to promote the transcription of Cped1. Dual-luciferase reporter assay revealed that active control area of the chicken Cped1 gene is −296 to −1 bp. There are Cebpb, Sp1, and Sox2 transcription factor binding sites in this region. Point-mutation experiment results showed that Sox2 negatively regulates the transcription of Cped1. Above results demonstrated that Cped1 is a key gene that regulates the formation of SSCs. Histone acetylation and transcription factor Sox2 participate in the regulation of Cped1.
Highlights
Spermatogonial stem cell (SSCs) is the only adult stem cell in the body that can transmit genetic information to their offspring. They have the potential to be induced to multiple cells in vitro, which may replace embryonic stem cells (ESC) for gene therapy and genetic research without ethical issues
In order to explore the key genes in the formation of chicken spermatogonial stem cell (SSC) and complete the regulation mechanism of SSCs, we found cadherin-like and PC-esterase domain containing 1 (Cped1) was expressed in SSCs by RNA sequencing (RNA-seq) of chicken ESCs, primordial germ cell (PGC), and SSCs [16]
Overexpression of Cped1 could not increase the amount of SSCs, it can improve the expression of germ cell marker genes at the transcription level (Figure 2B,D) These results predicted that knocking out Cped1 may inhibit the formation of PGCs, which indirectly affects the formation of SSCs
Summary
Spermatogonial stem cell (SSCs) is the only adult stem cell in the body that can transmit genetic information to their offspring. The problems are constantly highlighted: the surface markers are often not expressed genes in SSCs c 2018 The Author(s). Many researchers have focused on exploring gene regulation for SSCs self-renewal and formation at different levels. It is reasonable to speculate that Cped may play an important role in the formation of SSCs. In order to reveal the regulation of Cped in the formation of chicken SSCs, we knocked out and overexpressed Cped in vitro and in vivo.
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