Research Article

Single-cell reconstruction of developmental trajectories during zebrafish embryogenesis

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Science  01 Jun 2018:
Vol. 360, Issue 6392, eaar3131
DOI: 10.1126/science.aar3131

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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

Structured Abstract

INTRODUCTION

During embryogenesis, pluripotent cells gradually become specialized and acquire distinct functions and morphologies. Because much of the specification process is controlled through changes in gene expression, the identification of the transcriptional trajectories underlying cell fate acquisition is paramount to understanding and manipulating development.

RATIONALE

Traditional approaches have studied specific fate decisions by analyzing the transcription of a few selected marker genes or by profiling isolated, predefined cell populations. The advent of large-scale single-cell RNA sequencing (scRNA-seq) provides the means to comprehensively define the gene expression states of all embryonic cells as they acquire their fates. This technology raises the possibility of identifying the molecular trajectories that describe cell fate specification by sampling densely during embryogenesis and connecting the transcriptomes of cells that have similar gene expression profiles. However, the numerous transcriptional states and branch points, as well as the asynchrony in developmental processes, pose major challenges to the computational reconstruction of developmental trajectories from scRNA-seq data.

RESULTS

We generated single-cell transcriptomes from 38,731 cells during early zebrafish embryogenesis at high temporal resolution, spanning 12 stages from the onset of zygotic transcription through early somitogenesis. We took two complementary approaches to identify the transcriptional trajectories in the data. First, we developed a simulated diffusion-based computational approach, URD, which identified the trajectories describing the specification of 25 cell types in the form of a branching tree. Second, we identified modules of coexpressed genes and connected them across developmental time. Combining the reconstructed developmental trajectories with differential gene expression analysis uncovered gene expression cascades leading to each cell type, including previously unidentified markers and candidate regulators. Combining these trajectories with Seurat, which infers the spatial positions of cells on the basis of their transcriptomes, connected the earlier spatial position of progenitors to the later fate of their descendants.

Inspection of the developmental tree led to new insights about molecular specification in zebrafish. For example, the first branch point in the tree indicated that the first molecular specification event may not only separate the germ layers but also define the axial versus nonaxial mesendoderm. Additionally, some developmental branch points contained intermediate cells that expressed genes characteristic of multiple downstream cell fates. Gene expression analysis at one such branch point (the axial mesoderm) suggested that the intermediate cells switch their specification from one fate (notochord) to another (prechordal plate). Last, analysis of single-cell transcriptomes from a Nodal-signaling mutant revealed that even at the whole-transcriptome level, mutant cells were canalized into a subset of wild-type states and did not adopt any transcriptional states not observed in wild type, despite abnormal developmental signaling.

CONCLUSION

These findings reconstruct the gene expression trajectories during the embryogenesis of a vertebrate and highlight the concurrent canalization and plasticity of cell type specification. The scRNA-seq data and developmental tree provide a rich resource for future studies in zebrafish: The raw and processed data and the URD software are available for download, and the data can be browsed interactively online. Last, this approach provides a broadly applicable framework with which to reconstruct complex developmental trajectories from single-cell transcriptomes.

Developmental tree of early zebrafish embryogenesis.

Single-cell transcriptomes were generated from zebrafish embryos at 12 developmental stages (six of which are shown). The transcriptional trajectories that describe the fate specification of 25 cell types were reconstructed from the data. Molecular specification is visualized with a force-directed layout, in which each cell is represented by a point (colored by developmental stage), proceeding from pluripotent cells (at the bottom center) outward to 25 distinct cell types. A subset of the identified trajectories are labeled in groups.

Abstract

During embryogenesis, cells acquire distinct fates by transitioning through transcriptional states. To uncover these transcriptional trajectories during zebrafish embryogenesis, we sequenced 38,731 cells and developed URD, a simulated diffusion-based computational reconstruction method. URD identified the trajectories of 25 cell types through early somitogenesis, gene expression along them, and their spatial origin in the blastula. Analysis of Nodal signaling mutants revealed that their transcriptomes were canalized into a subset of wild-type transcriptional trajectories. Some wild-type developmental branch points contained cells that express genes characteristic of multiple fates. These cells appeared to trans-specify from one fate to another. These findings reconstruct the transcriptional trajectories of a vertebrate embryo, highlight the concurrent canalization and plasticity of embryonic specification, and provide a framework with which to reconstruct complex developmental trees from single-cell transcriptomes.

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