Caenorhabditis elegans is an animal with few cells but a wide diversity of cell types. In this study, we characterize the molecular basis for their specification by profiling the transcriptomes of 86,024 single embryonic cells. We identify 502 terminal and preterminal cell types, mapping most single-cell transcriptomes to their exact position in C. elegans’ invariant lineage. Using these annotations, we find that (i) the correlation between a cell’s lineage and its transcriptome increases from middle to late gastrulation, then falls substantially as cells in the nervous system and pharynx adopt their terminal fates; (ii) multilineage priming contributes to the differentiation of sister cells at dozens of lineage branches; and (iii) most distinct lineages that produce the same anatomical cell type converge to a homogenous transcriptomic state.

Highlights d Proximal nephron segments show distinct expression profiles between the sexes d The time of nephron formation determines position and segmental cell diversity d Lineage convergence is observed at nephron-collecting system junctions d Data can be queried and viewed within an annotated anatomical database

C. elegans is an animal with few cells, but a striking diversity of cell types. Here, we characterize the molecular basis for their specification by profiling the transcriptomes of 84,625 single embryonic cells. We identify 284 terminal and pre-terminal cell types, mapping most single cell transcriptomes to their exact position in C. elegans' invariant lineage. We use these annotations to perform the first quantitative analysis of the relationship between lineage and the transcriptome for a whole organism. We find that a strong lineage-transcriptome correlation in the early embryo breaks down in the final two cell divisions as cells adopt their terminal fates and that most distinct lineages that produce the same anatomical cell type converge to a homogenous transcriptomic state. Users can explore our data with a graphical application "VisCello". Main text:To understand how cell fates are specified during development, it is essential to know the temporal sequence of gene expression in cells during their trajectories from uncommitted precursors to differentiated terminal cell types. Gene expression patterns near branch points in these trajectories can help identify candidate regulators of cell fate decisions (1). Single cell RNA sequencing (sc-RNA-seq) has made it possible to obtain comprehensive measurements of Fig 3. Developmental trajectories of ciliated neurons. (A) UMAP of ciliated neurons and precursors.Colors correspond to cell identity. Text labels indicate terminal cells. Numbers 1-13 indicate parents of 1 ADE-ADA, 2 CEP-URX 3 IL1 4 OLL 5 OLQ 6 ASJ-AUA 7 ASE 8 ASI 9 ASK 10 ADF-AWB 11 ASG-AWA 12 ADL 13 AFD-RMD. 3-5, 7-9, and 12 are listed as parents of only one cell type as the sister cells die. Numbers 14-17 indicate grandparents of 14 IL1 (= IL2 parent) 15 OLQ-URY 16, 17 ASE-ASJ-AUA. 18 indicates a progenitor cluster that includes the AWC-SAAVx and BAG-SMDVx parents, which were identified in a separate UMAP (Fig. S12C). This latter analysis also tentatively identified a few cells near the base of the ASH trajectory as the ASH-RIB parent. Late stage AUA cells cluster with non-ciliated neurons and are not included in this UMAP but are included in the heatmap in panel D. The tiny cluster of cells labeled with an asterisk (*) is putatively AWC-ON based on srt-28 expression. (B) UMAP plot colored by embryo time (colors matched to Fig. 1A) and gene expression (red indicates >0 reads for the listed gene). mcm-7 is gene associated with the cell cycle. unc-130 is known to be expressed in the ASG-AWA neuroblast but neither terminal cell (40) (C) Cartoon illustrating the lineage of the ASE, ASJ, and AUA neurons. (D) Heatmap showing patterns of differential transcription factor expression associated with branches in the ASE-ASJ-AUA lineage. Expression values are log-transformed, then centered and scaled by standard deviation for each row (gene).

Hematopoietic stem and progenitor cells (HSPCs) in the bone marrow are derived from a small population of hemogenic endothelial (HE) cells located in the major arteries of the mammalian embryo. HE cells undergo an endothelial to hematopoietic cell transition (EHT), giving rise to HSPCs that accumulate in intra-arterial clusters (IAC) before colonizing the fetal liver. To examine the cell and molecular transitions between endothelial (E), HE, and IAC cells, and the heterogeneity of HSPCs within IACs, we profiled ~37,000 cells from the caudal arteries [dorsal aorta (DA), umbilical (U), vitelline (V)] of embryonic day 9.5 (E9.5) to E11.5 mouse embryos by single-cell RNA sequencing (scRNA-Seq) and single-cell assay for transposase-accessible chromatin sequencing (scATAC-Seq). We identified a continuous developmental trajectory from E to HE to IAC cells, with identifiable intermediate stages. The intermediate stage most proximal to HE, which we term pre-HE, is characterized by increased accessibility of chromatin enriched for SOX, FOX, GATA, and SMAD motifs. A developmental bottleneck separates pre-HE from HE, with RUNX1 dosage regulating the efficiency of the pre-HE to HE transition. A distal candidate Runx1 enhancer exhibits high chromatin accessibility specifically in pre-HE cells at the bottleneck, but loses accessibility thereafter. Distinct developmental trajectories within IAC cells result in two populations of CD45+ HSPCs; an initial wave of lympho-myeloid-biased progenitors, followed by precursors of hematopoietic stem cells (pre-HSCs). This multi-omics single-cell atlas significantly expands our understanding of pre-HSC ontogeny.

reports receiving commercial research grants from Kite, Servier, and Novartis, is listed as inventor on a patent for toxicity management for antitumor activity of CARs (WO 2014011984 A1; managed according to the University of Pennsylvania patent policy), and reports receiving other remuneration from McNaul Ebel. D.M.B. is now an employee of Tmunity Therapeutics, Inc. No potential conflicts of interest were disclosed by the other authors.Research.