Embryonic genome instability upon DNA replication timing program emergence

Faithful DNA replication is essential for genome integrity. Under-replicated DNA leads to chromosome segregation defects, which are reportedly common during embryogenesis. However, DNA replication regulation remains poorly understood in early mammalian embryos. Here, we constructed a single-cell genome-wide DNA replication atlas of pre-implantation mouse embryos and discovered an abrupt replication program switch accompanied by a transient period of genomic instability. In 1- and 2-cell embryos, we observed the complete absence of a replication timing (RT) program, and the entire genome replicated gradually and uniformly using extremely slow-moving forks. In 4-cell embryos, a somatic-cell-like RT program commenced abruptly. However, the fork speed was still slow, S-phase was extended, and SLX4 DNA repair foci increased during G2/M, which was followed by a transient increase in chromosome segregation errors. Importantly, live imaging captured longer S-phase of error cells, and the breakpoints identified by single-cell genome sequencing were enriched in late-replicating regions. By the 8-cell stage, forks gained speed, S-phase was no longer extended, and chromosome aberrations disappeared. Thus, a transient period of genomic instability exists during normal mouse development, which is preceded by a fragile S-phase lacking the coordination between replisome-level regulation and megabase-scale RT regulation, implicating the importance of their coordination for genome integrity. Overall design: scRepli-seq experiments were performed using the embryos of the following mouse strains, conditions and the developmental stages (single cells were collected by the micromanipulation): (1) In-vitro developed embryos from the BDF1 strain including the developmental stages of 4-cell, 8-cell, 16-cell, ICM (inner cell mass), and TE (trophectoderm). Titles are: "BDF1_in-vitro_(developmental stage)_(project ID)" (2) In-vitro developed embryos by IVF (in-vitro fertilization) or ISCI (intracytoplasmic sperm injection) using the B6MSM strain (an F1-hybrid between B6 female and MSM male) by including the developmental stages of 1-cell, 2-cell, and 4-cell. Titles are: "B6MSM_(developmental stage)_(project ID)" (3) SCNT (somatic cell nuclear transfer, cumulus cells as donors) embryos using the BDF1 strain including the developmental stages of 1-cell, 2-cell. Titles are: "SCNT_(developmental stage)_(project ID)" (4) EdU labeled & stained embryos from the B6 strain including the developmental stages of 4-cell and 8-cell. Titles are: "B6_EdU_(developmental stage)_(project ID)" (5) In-vitro developed embryos from BDF1 strain collected mainly in the G1 phase, including the developmental stages of 4-cell and 8-cell. Titles are: "BDF1_in-vitro_G1_(developmental stage)_(project ID)" (6) In-vivo developed embryos from the oviduct of the mouse (BDF1 strain) including the developmental stages of 4-cell, 8-cell, and 16-cell. Titles are: "BDF1_in-vivo_(developmental stage)_(project ID)" Project IDs are "DEP_01_XX", "P445_XX", or "LCS_01_XX" Metadata for each single-cell sample can be found in the supplementary file "scRT_metadata.txt.gz"