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Single-Cell Transcriptomes Reveal Cell Differentiation Dynamics in Zebrafish, Frog Embyogenesis

NEW YORK (GenomeWeb) – A set of zebrafish and Xenopus frog single-cell studies published online today in Science is revealing previously unappreciated details on vertebrate embryogenesis.

In one of these papers, a Harvard University- and Broad Institute-led team outlined a computational method dubbed URD that it came up with to reconstruct zebrafish developmental trajectories based on single-cell RNA sequences for 38,731 individual cells plucked from nearly 700 embryos. 

The researchers used a single-cell RNA-seq strategy called Drop-seq to profile the 694 zebrafish embryos, ranging in age from just over three hours post-fertilization to a far more advanced developmental stage 12 hours after fertilization, when many cell types have already undergone differentiation. 

Using these data, coupled with their URD simulated diffusion-based computational tool, they successfully followed the expression dynamics in more than two dozen cell types over the 12 development stages considered. In the process, they pinned down gene sets expressed together at specific developmental stages, for example, while characterizing the ever-growing transcriptomic chasms cells differentiating into distinct tissue types.

For a related analysis, the team turned to deeper single-cell RNA-seq data generated with the SMART-Seq2 protocol to distinguish between cells from wild-type zebrafish and zebrafish mutants missing a gene involved in the formation of mesendoderm tissue. Though differences did arise in the mutant, the results also highlighted plasticity in the embryo, coupled with relative developmental stability despite mutation, providing a look at the phenotypic breadth and boundaries associated with each cell type (known as canalization).

"These findings reconstruct the transcriptional trajectories of a vertebrate embryo, highlight the concurrent canalization and plasticity of embryonic specification, and provide a framework to reconstruct complex developmental trees from single-cell transcriptomes," co-corresponding authors Alexander Schier and Aviv Regev, both affiliated with the Broad Institute, and their colleagues wrote.

For their own Science study, researchers from Harvard Medical School began by using a single-cell RNA-seq method called inDrops to characterize more than 92,000 zebrafish embryo transcripts, representing developmental stages spanning the first day of development.

"From this dataset, clustering of the wild-type transcriptomes revealed an expanding set of epidermal, neural, mesodermal, and endodermal cell states over developmental time, many of which could be specifically annotated based on expression of marker genes," senior authors Allon Klein and Sean Megason, systems biology researchers at Harvard, wrote with their co-authors.

That team turned to an unbiased graph-based analytical method to bring these data together and chart cell trajectories over time and space in the developing zebrafish, exploring everything from cell type hierarchies and connections to canalization.

The researchers took a closer look at individual cell routes in the zebrafish embryo with an approach called "sequencing of transcribed clonally encoded random barcodes" (TracerSeq), a transposon-based barcode sequencing strategy to follow individual cells and clonal dynamics in the embryos.

Using CRISPR-Cas9 gene editing to lop out part of the chordin locus, they also used single-cell RNA-seq to see how embryogenesis shifts in the absence of the documented developmental gene.

"We anticipate that the synthesis of single-cell lineage and transcriptome information will continue to be crucial for deciphering how cells traverse state trajectories with complex topologies," the authors wrote, noting that "[s]ingle-cell mapping of genetic perturbation data presents a powerful framework for identifying regulatory features of a developmental landscape."

Klein and Harvard systems biology researcher Marc Kirschner led a team that published a related analysis of cell differentiation dynamics in developing Western claw-toed frog (Xenopus tropicalis) embryos.

Using inDrop single-cell RNA-seq, the investigators profiled cell trajectories for nearly 137,000 individual cells representing 10 developmental stages in the frog — results they compared with findings from the developing zebrafish.

"We find that many embryonic cell states appear earlier than previously appreciated," the authors reported, adding that the developing zebrafish comparison pointed to "conserved and divergent features of vertebrate early developmental gene expression programs."