Integrating ATAC-Seq and RNA-Seq Reveals the Signal Regulation Involved in the Artemia Embryonic Reactivation Process

Abstract

Embryonic diapause is a common evolutionary adaptation observed across a wide range of organisms. Artemia is one of the classic animal models for diapause research. The current studies of Artemia diapause mainly focus on the induction and maintenance of the embryonic diapause, with little research on the molecular regulatory mechanism of Artemia embryonic reactivation. The first 5 h after embryonic diapause breaking has been proved to be most important for embryonic reactivation in Artemia. In this work, two high-throughput sequencing methods, ATAC-seq and RNA-seq, were integrated to study the signal regulation process in embryonic reactivation of Artemia at 5 h after diapause breaking. Through the GO and KEGG enrichment analysis of the high-throughput datasets, it was showed that after 5 h of diapause breaking, the metabolism and regulation of Artemia cyst were quite active. Several signal transduction pathways were identified in the embryonic reactivation process, such as G-protein-coupled receptor (GPCR) signaling pathway, cell surface receptor signaling pathway, hormone-mediated signaling pathway, Wnt, Notch, mTOR signaling pathways, etc. It indicates that embryonic reactivation is a complex process regulated by multiple signaling pathways. With the further protein structure analysis and RT-qPCR verification, 11 GPCR genes were identified, in which 5 genes function in the embryonic reactivation stage and the other 6 genes contribute to the diapause stage. The results of this work reveal the signal transduction pathways and GPCRs involved in the embryonic reactivation process of Artemia cysts. These findings offer significant clues for in-depth research on the signal regulatory mechanisms of the embryonic reactivation process and valuable insights into the mechanism of animal embryonic diapause.

Keywords: Artemia cyst; G-protein-coupled receptor; embryonic diapause; embryonic reactivation; signal transduction.

Figure 1

The lifespan of Artemia.

Figure 2

The volcano plot and genome-wide distribution of differential enriched peaks from ATAC-seq. (a) The volcano plot of differential enriched peaks. (b) The genome-wide distribution of differential enriched peaks.

Figure 3

GO enrichment of DEGs from ATAC-seq.

Figure 4

The volcano plot of DEGs from RNA-seq.

Figure 5

GO enrichment of DEGs from RNA-seq.

Figure 6

GO and KEGG enrichment analysis of integrated DEGs.

Figure 7

Enrichment of DEGs in the childhood of “regulation of biological process”.

Figure 8

Results of RT-qPCR for the candidate genes. ‘**’ at 0.01 level. ‘***’ at 0.001 level, ‘****’ at 0.0001 level.

Figure 9

Comparison of the results among “0–30 min”, “30 min–5 h”, and “0–5 h” groups. (a) Comparison results of DEGs. (b) Comparison results of GPCR genes. The up-regulated GPCR genes were indicated in red, and the down-regulated GPCR genes were indicated in green.