Daniel E. Wagner

Clonogenic neoblasts are pluripotent adult stem cells that underlie planarian regeneration

A pluripotent adult stem cell underlies flatworms’ amazing regenerative ability. Pluripotent cells in the embryo can generate all cell types, but lineage-restricted cells are generally thought to replenish adult tissues. Planarians are flatworms and regenerate from tiny body fragments, a process requiring a population of proliferating cells (neoblasts). Whether regeneration is accomplished by pluripotent cells or by the collective activity of multiple lineage-restricted cell types is unknown. We used ionizing radiation and single-cell transplantation to identify neoblasts that can form large descendant-cell colonies in vivo. These clonogenic neoblasts (cNeoblasts) produce cells that differentiate into neuronal, intestinal, and other known postmitotic cell types and are distributed throughout the body. Single transplanted cNeoblasts restored regeneration in lethally irradiated hosts. We conclude that broadly distributed, adult pluripotent stem cells underlie the remarkable regenerative abilities of planarians.

Genetic regulators of a pluripotent adult stem cell system in planarians identified by RNAi and clonal analysis

Pluripotency is a central, well-studied feature of embryonic development, but the role of pluripotent cell regulation in somatic tissue regeneration remains poorly understood. In planarians, regeneration of entire animals from tissue fragments is promoted by the activity of adult pluripotent stem cells (cNeoblasts). We utilized transcriptional profiling to identify planarian genes expressed in adult proliferating, regenerative cells (neoblasts). We also developed quantitative clonal analysis methods for expansion and differentiation of cNeoblast descendants that, together with RNAi, revealed gene roles in stem cell biology. Genes encoding two zinc finger proteins, Vasa, a LIM domain protein, Sox and Jun-like transcription factors, two candidate RNA-binding proteins, a Setd8-like protein, and PRC2 (Polycomb) were required for proliferative expansion and/or differentiation of cNeoblast-derived clones. These findings suggest that planarian stem cells utilize molecular mechanisms found in germ cells and other pluripotent cell types and identify genetic regulators of the planarian stem cell system.

Simultaneous single-cell profiling of lineages and cell types in the vertebrate brain.

The lineage relationships among the hundreds of cell types generated during development are difficult to reconstruct. A recent method, GESTALT, used CRISPR–Cas9 barcode editing for large-scale lineage tracing, but was restricted to early development and did not identify cell types. Here we present scGESTALT, which combines the lineage recording capabilities of GESTALT with cell-type identification by single-cell RNA sequencing. The method relies on an inducible system that enables barcodes to be edited at multiple time points, capturing lineage information from later stages of development. Sequencing of ∼60,000 transcriptomes from the juvenile zebrafish brain identified >100 cell types and marker genes. Using these data, we generate lineage trees with hundreds of branches that help uncover restrictions at the level of cell types, brain regions, and gene expression cascades during differentiation. scGESTALT can be applied to other multicellular organisms to simultaneously characterize molecular identities and lineage histories of thousands of cells during development and disease.

Single-cell mapping of gene expression landscapes and lineage in the zebrafish embryo

Mapping the vertebrate developmental landscape As embryos develop, numerous cell types with distinct functions and morphologies arise from pluripotent cells. Three research groups have used single-cell RNA sequencing to analyze the transcriptional changes accompanying development of vertebrate embryos (see the Perspective by Harland). Wagner et al. sequenced the transcriptomes of more than 90,000 cells throughout zebrafish development to reveal how cells differentiate during axis patterning, germ layer formation, and early organogenesis. Farrell et al. profiled the transcriptomes of tens of thousands of embryonic cells and applied a computational approach to construct a branching tree describing the transcriptional trajectories that lead to 25 distinct zebrafish cell types. The branching tree revealed how cells change their gene expression as they become more and more specialized. Briggs et al. examined whole frog embryos, spanning zygotic genome activation through early organogenesis, to map cell states and differentiation across all cell lineages over time. These data and approaches pave the way for the comprehensive reconstruction of transcriptional trajectories during development. Science, this issue p. 981, p. eaar3131, p. eaar5780; see also p. 967 Single-cell RNA sequencing reveals cell type trajectories and cell lineage in the developing zebrafish embryo. High-throughput mapping of cellular differentiation hierarchies from single-cell data promises to empower systematic interrogations of vertebrate development and disease. Here we applied single-cell RNA sequencing to >92,000 cells from zebrafish embryos during the first day of development. Using a graph-based approach, we mapped a cell-state landscape that describes axis patterning, germ layer formation, and organogenesis. We tested how clonally related cells traverse this landscape by developing a transposon-based barcoding approach (TracerSeq) for reconstructing single-cell lineage histories. Clonally related cells were often restricted by the state landscape, including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in embryos lacking the chordin gene. We provide web-based resources for further analysis of the single-cell data.

The dynamics of gene expression in vertebrate embryogenesis at single-cell resolution

Mapping the vertebrate developmental landscape As embryos develop, numerous cell types with distinct functions and morphologies arise from pluripotent cells. Three research groups have used single-cell RNA sequencing to analyze the transcriptional changes accompanying development of vertebrate embryos (see the Perspective by Harland). Wagner et al. sequenced the transcriptomes of more than 90,000 cells throughout zebrafish development to reveal how cells differentiate during axis patterning, germ layer formation, and early organogenesis. Farrell et al. profiled the transcriptomes of tens of thousands of embryonic cells and applied a computational approach to construct a branching tree describing the transcriptional trajectories that lead to 25 distinct zebrafish cell types. The branching tree revealed how cells change their gene expression as they become more and more specialized. Briggs et al. examined whole frog embryos, spanning zygotic genome activation through early organogenesis, to map cell states and differentiation across all cell lineages over time. These data and approaches pave the way for the comprehensive reconstruction of transcriptional trajectories during development. Science, this issue p. 981, p. eaar3131, p. eaar5780; see also p. 967 A single-cell transcriptome analysis of whole frog embryos reveals cell states and provides a map of differentiation over time. INTRODUCTION Metazoan development represents a big jump in complexity compared with unicellular life in two aspects: cell-type differentiation and cell spatial organization. In vertebrate embryos, many distinct cell types appear within just a single day of life after fertilization. Studying the developmental dynamics of all embryonic cell types is complicated by factors such as the speed of early development, complex cellular spatial organization, and scarcity of raw material for conventional analysis. Genetics and experimental embryology have clarified major transcription factors and secreted signaling molecules involved in the specification of early lineages. However, development involves parallel alterations in many cellular circuits, not just a few well-described factors. RATIONALE We recently developed a microfluidics-based single-cell RNA sequencing method capable of efficiently profiling tens of thousands of individual transcriptomes. Building on earlier studies that showed how single-cell transcriptomics can reveal cell states within complex tissues, we reasoned that a series of such measurements from embryos, if collected with sufficient time resolution, could allow reconstruction of developmental cell-state hierarchies. We focused on the western claw-toed frog, Xenopus tropicalis, which serves as one of the best-studied model systems of early vertebrate development. We profiled these embryos from just before the onset of zygotic transcription up to a point at which dozens of distinct cell types have formed encompassing progenitors of most major organs. To establish aspects of development general to vertebrates, we additionally incorporated data from the copublished paper by Wagner et al. on zebrafish embryos, which separated from frogs about 400 million years ago. RESULTS We profiled 136,966 single-cell transcriptomes over the first day of life of Xenopus tropicalis. Our analysis classifies 259 gene expression clusters across 10 time points, which belong to 69 annotated embryonic cell types and capture further substructure. Using a computational approach to link cell states between time points, a resulting cell-state graph agrees well with previous lineage-tracing studies and shows that developmental fate choices can be well approximated by a treelike model. Many cell states are detected considerably earlier than previously understood, thus revealing the earliest events in their differentiation. The data lends clarity to numerous specific developmental processes, such as the developmental origin of the vertebrate neural crest. Through an evolutionary comparison with zebrafish, we identified diverging features of developmental dynamics, including many genes showing cell-type specificity in one organism but not in another. Yet, we also identified conserved patterns in the reuse of transcription factors across lineages and in multilineage priming at fate branch points. The resulting resource is available in an interactive online browser that allows in silico exploration of any gene in any cell state (tinyurl.com/scXen2018). CONCLUSION The approaches and results presented here, along with the copublished paper by Wagner et al., establish the first steps toward a data-driven dissection of developmental dynamics at the scale of entire organisms. They provide a useful, annotated resource for developmental biologists, comprehensively tracking differentiation programs as they unfold on a high-dimensional gene expression landscape. Although demonstrated on model organisms, the same approaches could be transformative to the study of nonmodel organisms by allowing rapid and quantitative description of differentiation processes across the tree of life, opening up a new front in evolutionary biology. Single-cell analysis of whole developing vertebrate embryos. Xenopus embryos at 10 time points over the first day of life were dissociated, barcoded, and sequenced, yielding 136,966 single-cell transcriptomes. These data were clustered and connected over time to reveal a complete view of transcriptional changes in each embryonic lineage and clarify numerous features of early development. hpf, hours postfertilization. Time series of single-cell transcriptome measurements can reveal dynamic features of cell differentiation pathways. From measurements of whole frog embryos spanning zygotic genome activation through early organogenesis, we derived a detailed catalog of cell states in vertebrate development and a map of differentiation across all lineages over time. The inferred map recapitulates most if not all developmental relationships and associates new regulators and marker genes with each cell state. We find that many embryonic cell states appear earlier than previously appreciated. We also assess conflicting models of neural crest development. Incorporating a matched time series of zebrafish development from a companion paper, we reveal conserved and divergent features of vertebrate early developmental gene expression programs.

teashirt is required for head-versus-tail regeneration polarity in planarians

Regeneration requires that the identities of new cells are properly specified to replace missing tissues. The Wnt signaling pathway serves a central role in specifying posterior cell fates during planarian regeneration. We identified a gene encoding a homolog of the Teashirt family of zinc-finger proteins in the planarian Schmidtea mediterranea to be a target of Wnt signaling in intact animals and at posterior-facing wounds. Inhibition of Smed-teashirt (teashirt) by RNA interference (RNAi) resulted in the regeneration of heads in place of tails, a phenotype previously observed with RNAi of the Wnt pathway genes β-catenin-1, wnt1, Dvl-1/2 or wntless. teashirt was required for β-catenin-1-dependent activation of posterior genes during regeneration. These findings identify teashirt as a transcriptional target of Wnt signaling required for Wnt-mediated specification of posterior blastemas. Summary: The zinc finger protein teashirt is a transcriptional target of Wnt signaling and is required for Wnt-mediated specification of cell fate during regeneration in planarians.

Simultaneous single-cell profiling of lineages and cell types in the vertebrate brain by scGESTALT

Hundreds of cell types are generated during development, but their lineage relationships are largely elusive. Here we report a technology, scGESTALT, which combines cell type identification by single-cell RNA sequencing with lineage recording by cumulative barcode editing. We sequenced ~60,000 transcriptomes from the juvenile zebrafish brain and identified more than 100 cell types and marker genes. We engineered an inducible system that combines early and late barcode editing and isolated thousands of single-cell transcriptomes and their associated barcodes. The large diversity of edited barcodes and cell types enabled the generation of lineage trees with hundreds of branches. Inspection of lineage trajectories identified restrictions at the level of cell types and brain regions and helped uncover gene expression cascades during differentiation. These results establish scGESTALT as a new and widely applicable tool to simultaneously characterize the molecular identities and lineage histories of thousands of cells during development and disease.

Genetic screening enters the single-cell era

Four studies overcome the limitations of pooled and arrayed genetic screens by integrating single-cell transcriptomics.