Michael V. Zuccaro

Mammalian species exist as diploid organisms, with only gametes existing as stable, nonproliferating haploids. Haploid mammalian pluripotent stem cells, in all derived species to date - mouse, human and rat - undergo spontaneous diploidization. Here, we investigated the mechanisms affecting the stability of the haploid state using the mouse and human embryo, as well as a human embryonic stem cells. We demonstrate that diploidization occurs early in preimplantation development and is often unproductive, with haploid embryos exhibiting decreased developmental potential. Haploid embryos show increased chromosome segregation errors at the first mitosis, failure to align chromosomes on the metaphase plate in the second mitosis. Delayed mitotic progression and failure to form a central spindle and a midbody are followed by cytokinesis failure, diploidization, increased DNA damage marked by γH2AX and RPA32 foci, and low developmental potential. By increasing the nucleo-cytoplasmic ratio in diploids, or reducing this ratio in haploids, these phenotypes can be induced in diploids or reduced in haploids. Changing ploidy alone from haploid to diploid without also adjusting nucleo-cytoplasmic ratio does not improve mitotic phenotypes and developmental outcomes. Thus, the most upstream driver responsible for the instability of the haploid state is a stage- specific ratio of nucleus to cell size.

DNA replication in differentiated cells follows a defined program, but when and how it is established during mammalian development is not known. Here we show using single-cell sequencing, that late replicating regions are established in association with the B compartment and the nuclear lamina from the first cell cycle after fertilization on both maternal and paternal genomes. Late replicating regions contain a relative paucity of active origins and few but long genes and low G/C content. In both bovine and mouse embryos, replication timing patterns are established prior to embryonic genome activation. Chromosome breaks, which form spontaneously in bovine embryos at sites concordant with human embryos, preferentially locate to late replicating regions. In mice, late replicating regions show enhanced fragility due to a sparsity of dormant origins that can be activated under conditions of replication stress. This pattern predisposes regions with long neuronal genes to fragility and genetic change prior to separation of soma and germ cell lineages. Our studies show that the segregation of early and late replicating regions is among the first layers of genome organization established after fertilization.

DNA replication in differentiated cells follows a defined program, but when and how it is established during mammalian development is not known. Here we show using single-cell sequencing, that both bovine and mouse cleavage stage embryos progress through S-phase in a defined pattern. Late replicating regions are associated with the nuclear lamina from the first cell cycle after fertilization, and contain few active origins, and few but long genes. Chromosome breaks, which form spontaneously in bovine embryos at sites concordant with human embryos, preferentially locate to late replicating regions. In mice, late replicating regions show enhanced fragility due to a sparsity of dormant origins that can be activated under conditions of replication stress. This pattern predisposes regions with long neuronal genes to fragility and genetic change prior to segregation of soma and germ line. Our studies show that the formation of early and late replicating regions is among the first layers of epigenetic regulation established on the mammalian genome after fertilization.

Limitations in cell proliferation are important for normal function of differentiated tissues and essential for the safety of cell replacement products made from pluripotent stem cells, which have unlimited proliferative potential. To evaluate whether these limitations can be established pharmacologically, we exposed pancreatic progenitors differentiating from human pluripotent stem cells to small molecules that interfere with cell cycle progression either by inducing G ₁ arrest or by impairing S phase entry or S phase completion and determined growth potential, differentiation, and function of insulin-producing endocrine cells. We found that the combination of G ₁ arrest with a compromised ability to complete DNA replication promoted the differentiation of pancreatic progenitor cells toward insulin-producing cells and could substitute for endocrine differentiation factors. Reduced replication fork speed during differentiation improved the stability of insulin expression, and the resulting cells protected mice from diabetes without the formation of cystic growths. The proliferative potential of grafts was proportional to the reduction of replication fork speed during pancreatic differentiation. Therefore, a compromised ability to enter and complete S phase is a functionally important property of pancreatic endocrine differentiation, can be achieved by reducing replication fork speed, and is an important determinant of cell-intrinsic limitations of growth.

Human cleavage stage embryos frequently acquire chromosomal aneuploidies during mitosis. We recently showed that double strand breaks (DSBs) introduced by Cas9 result in frequent chromosomal alterations. Here we show that spontaneous DSBs arise de novo during embryonic DNA replication and show as chromosomal breaks that occur primarily, in centromeric, and gene-poor areas, with the latter harboring long genes implicated in neurodevelopmental disorders, including CNTN5, IMMP2L, LRP1B, RYR2, CSMD1. While transcription-replication conflicts are not required for chromosome fragility, fragile, gene-poor regions contain long-traveling replication forks, and complete replication late in G2 phase, hours after most of the genome has been duplicated. Incomplete replication at these sites in both human and mouse zygotes, results in DSBs, whole and segmental chromosome errors, micronucleation, chromosome fragmentation and poor embryo quality. Thus, failure to complete DNA replication is a mechanism of developmental failure, and a source of detrimental genetic alterations in humans.

Reprogrammed pluripotent stem cells (PSCs) are valuable for research and potentially for cell replacement therapy. However, only a fraction of reprogrammed PSCs are developmentally competent. Genomic stability and accurate DNA synthesis are fundamental for cell development and critical for safety. We analyzed whether defects in DNA replication contribute to genomic instability and the diverse differentiation potentials of reprogrammed PSCs. Using a unique single-molecule approach, we visualized DNA replication in isogenic PSCs generated by different reprogramming approaches, either somatic cell nuclear transfer (NT-hESCs) or with defined factors (iPSCs). In PSCs with lower differentiation potential, DNA replication was incompletely reprogrammed, and genomic instability increased during replicative stress. Reprogramming of DNA replication did not correlate with DNA methylation. Instead, fewer replication origins and a higher frequency of DNA breaks in PSCs with incompletely reprogrammed DNA replication were found. Given the impact of error-free DNA synthesis on the genomic integrity and differentiation proficiency of PSCs, analyzing DNA replication may be a useful quality control tool.

Summary: The correction of disease-causing mutations in human embryos could reduce the burden of inherited genetic disorders in the fetus and newborn, and improve the efficiency of fertility treatments for couples with disease-causing mutations in lieu of embryo selection. Here we evaluate the repair outcomes of a Cas9-induced double-strand break (DSB) introduced on the paternal chromosome at the EYS locus, which carries a frame-shift mutation causing blindness. We show that the most common repair outcome is microhomology-mediated end joining, which occurs during the first cell cycle in the zygote, leading to embryos with non-mosaic restoration of the reading frame. However, about half of the breaks remain unrepaired, resulting in an undetectable paternal allele and, upon entry into mitosis, loss of one or both chromosomal arms. Thus, Cas9 allows for the modification of chromosomal content in human embryos in a targeted manner, which may be useful for the prevention of trisomies. Highlights: Cas9-mediated DSB induction and repair by end joining occurs within hours End joining provides an efficient way to restore reading frames without mosaicism Unrepaired DSBs persist through mitosis and result in frequent chromosome loss