A key goal of developmental biology is to understand how a single cell transforms into a full-grown organism comprising many different cell types. Single-cell RNA-sequencing (scRNA-seq) is commonly used to identify cell types in a tissue or organ1. However, organizing the resulting taxonomy of cell types into lineage trees to understand developmental origin of cells remains challenging. Here we present LINNAEUS (LINeage tracing by Nuclease-Activated Editing of Ubiquitous Sequences)—a strategy for simultaneous lineage tracing and transcriptome profiling in thousands of single cells. By combining scRNA-seq with computational analysis of lineage barcodes, generated by genome editing of transgenic reporter genes, we reconstruct developmental lineage trees in zebrafish larvae, and in heart, liver, pancreas and telencephalon of adult fish. LINNAEUS provides a systematic approach for tracing the origin of novel cell types, or known cell types under different conditions.

Chromosomes are folded into highly compacted structures to accommodate physical constraints within nuclei and to regulate access to genomic information. Recently, global mapping of pairwise contacts showed that loops anchoring topological domains (TADs) are highly conserved between cell types and species. Whether pairwise loops synergize to form higher-order structures is still unclear. Here we develop a conformation capture assay to study higher-order organization using chromosomal walks (C-walks) that link multiple genomic loci together into proximity chains in human and mouse cells. This approach captures chromosomal structure at varying scales. Inter-chromosomal contacts constitute only 7-10% of the pairs and are restricted by interfacing TADs. About half of the C-walks stay within one chromosome, and almost half of those are restricted to intra-TAD spaces. C-walks that couple 2-4 TADs indicate stochastic associations between transcriptionally active, early replicating loci. Targeted analysis of thousands of 3-walks anchored at highly expressed genes support pairwise, rather than hub-like, chromosomal topology at active loci. Polycomb-repressed Hox domains are shown by the same approach to enrich for synergistic hubs. Together, the data indicate that chromosomal territories, TADs, and intra-TAD loops are primarily driven by nested, possibly dynamic, pairwise contacts.

We developed a targeted chromosome conformation capture (4C) approach that uses unique molecular identifiers (UMIs) to derive high-complexity quantitative chromosome contact profiles with controlled signal-to-noise ratios. UMI-4C detects chromosomal interactions with improved sensitivity and specificity, and it can easily be multiplexed to allow robust comparison of contact distributions between loci and conditions. This approach may open the way to the incorporation of contact distributions into quantitative models of gene regulation.

DNA synthesis must be performed with extreme precision to maintain genomic integrity. In mammalian cells, different genomic regions are replicated at defined times, perhaps to preserve epigenetic information and cell differentiation status. However, the molecular principles that define this S phase program are unknown. By analyzing replication foci within discrete chromosome territories during interphase, we show that foci which are active during consecutive intervals of S phase are maintained as spatially adjacent neighbors throughout the cell cycle. Using extended DNA fibers, we demonstrate that this spatial continuity of replication foci correlates with the genetic continuity of adjacent replicon clusters along chromosomes. Finally, we used bioinformatic tools to compare the structure of DNA foci with DNA domains that are seen to replicate during discrete time intervals of S phase using genome-wide strategies. Data presented show that a major mechanism of S phase progression involves the sequential synthesis of regions of the genome because of their genetic continuity along the chromosomal fiber.

The control of DNA replication is of fundamental importance as cell proliferation demands that identical copies of the genetic material are passed to the two daughter cells that form during mitosis. These genetic copies are generated in the preceding S phase, where the entire DNA complement of the mother cell must be copied exactly once. As part of this process, it is known that different regions of mammalian genomes are replicated at specific times of a temporally defined replication programme. The key feature of this programme is that active genes in euchromatin are replicated before inactive ones in heterochromatin. This separation of S phase into periods where different classes of chromatin are duplicated is important in maintaining changes in gene expression that define individual cell types. Recent attempts to understand the structure of the S-phase timing programme have focused on the use of genome-wide strategies that inevitably use DNA isolated from large cell populations for analysis. However, this approach provides a composite view of events that occur within a population without knowledge of the cell-to-cell variability across the population. In this review, we attempt to combine information generated using genome-wide and single cell strategies in order to develop a coherent molecular understanding of S-phase progression. During this integration, we have explored how available information can be introduced into a modelling environment that best describes S-phase progression in mammalian cells.