Abstract

DNA is susceptible to damage by various sources. When the DNA is damaged, the cell repairs the damage through an appropriate DNA repair pathway. When the cell fails to repair DNA damage, apoptosis is initiated. Although several genes are involved in five major DNA repair pathways and two major apoptosis pathways, a comprehensive understanding of those gene expression is not well-understood in chicken tissues. We performed whole-transcriptome sequencing (WTS) analysis in the chicken embryonic fibroblasts (CEFs), stage X blastoderms, and primordial germ cells (PGCs) to uncover this deficiency. Stage X blastoderms mostly consist of undifferentiated progenitor (pluripotent) cells that have the potency to differentiate into all cell types. PGCs are also undifferentiated progenitor cells that later differentiate into male and female germ cells. CEFs are differentiated and abundant somatic cells. Through WTS analysis, we identified that the DNA repair pathway genes were expressed more highly in blastoderms and high in PGCs than CEFs. Besides, the apoptosis pathway genes were expressed low in blastoderms and PGCs than CEFs. We have also examined the WTS-based expression profiling of candidate pluripotency regulating genes due to the conserved properties of blastoderms and PGCs. In the results, a limited number of pluripotency genes, especially the core transcriptional network, were detected higher in both blastoderms and PGCs than CEFs. Next, we treated the CEFs, blastoderm cells, and PGCs with hydrogen peroxide (H2O2) for 1 h to induce DNA damage. Then, the H2O2 treated cells were incubated in fresh media for 3–12 h to observe DNA repair. Subsequent analyses in treated cells found that blastoderm cells and PGCs were more likely to undergo apoptosis along with the loss of pluripotency and less likely to undergo DNA repair, contrasting with CEFs. These properties of blastoderms and PGCs should be necessary to preserve genome stability during the development of early embryos and germ cells, respectively.

Highlights

  • DNA is susceptible to damage by various sources

  • The Eyal-Giladi and Kochav (EGK).X blastoderm is composed of mostly undifferentiated progenitor cells with the potency to differentiate into all cell types, and blastoderm-derived chicken embryonic stem cells (ESCs) have been established in vitro[5,6]

  • Based on the total quantified genes (24,356), the relationships between chicken embryonic fibroblasts (CEFs), EGK.X blastoderms, and primordial germ cells (PGCs) were investigated through several approaches

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Summary

Introduction

DNA is susceptible to damage by various sources. When the DNA is damaged, the cell repairs the damage through an appropriate DNA repair pathway. We performed whole-transcriptome sequencing (WTS) analysis in the chicken embryonic fibroblasts (CEFs), stage X blastoderms, and primordial germ cells (PGCs) to uncover this deficiency. Stage X blastoderms mostly consist of undifferentiated progenitor (pluripotent) cells that have the potency to differentiate into all cell types. We performed whole-transcriptome sequencing (WTS)-based expression profiling of DNA repair pathway, apoptosis pathway, and pluripotency regulating genes in EGK.X blastoderms and PGCs (target samples) compared with CEFs (reference sample). Of these pathways: BER, NER, and MMR repairs single-strand break; BER and NER repairs base damage; NHEJ repairs double-strand break, with errors; HR repairs double-strand break error-free; and all pathway correct non-double-strand break clustered DNA l­esions[18,19,22] These pathways are critical for maintaining the genome stability of cells. Both pathways lead to the final activation of caspase-3 and caspase-7, which cleave over 1000 proteins leading to their degradation or a­ ctivation[23]

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