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.

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).

The sea anemone Nematostella vectensis is the leading developmental and genomic model for the phylum Cnidaria, which includes anemones, hydras, jellyfish, and corals. In insects and vertebrates, the NF-B pathway is required for cellular and organismal responses to various stresses, including pathogens and chemicals, as well as for several developmental processes. Herein, we have characterized proteins that comprise the core NF-B pathway in Nematostella, including homologs of NF-B, IB, Bcl-3, and IB kinase (IKK).

Investigation of human CNS disease and drug effects has been hampered by the lack of a system that enables single cell analysis on live adult patient brain cells. We developed a culturing system, based on a papain-aided procedure, for resected adult human brain tissue removed during neurosurgery. We performed single-cell transcriptomics on over 300 cells permitting identification of oligodendrocytes, microglia, neurons, endothelial cells, and astrocytes after 3 weeks in culture. Using deep sequencing, we detected over 12,000 expressed genes including hundreds of cell-type enriched mRNAs, lncRNAs and pri-miRNAs. We describe cell-type and patient specific transcriptional hierarchies. Single-cell transcriptomics on cultured live adult patient derived cells is a prime example of the promise of personalized precision medicine. As these cells derive from subjects ranging in age into their sixties, this system permits human aging studies previously possible only in rodent systems.

Although regeneration is widespread among metazoa, the molecular mechanisms have been studied in only a handful of taxa. Of these taxa, fewer still are amenable to studies of embryogenesis. Our understanding of the evolution of regeneration, and its relation to embryogenesis, therefore remains limited. Using β-catenin as a marker, we investigated the role of canonical Wnt signaling during both regeneration and embryogenesis in the cnidarian Nematostella vectensis. The canonical Wnt signaling pathway is known to play a conserved role in primary axis patterning in triploblasts. Induction of Wnt signaling with alsterpaullone results in ectopic oral tissue during both regeneration and embryogenesis by specifically upregulating β-catenin expression, as measured by qRTPCR. Our data indicate that canonical Wnt signaling is sufficient for oral patterning during Nematostella regeneration and embryogenesis. These data also contribute to a growing body of literature indicating a conserved role for patterning mechanisms across various developmental modes of metazoans.