Research Article

The Drosophila embryo at single-cell transcriptome resolution

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Science  13 Oct 2017:
Vol. 358, Issue 6360, pp. 194-199
DOI: 10.1126/science.aan3235
  • Fig. 1 Deconstructing and reconstructing the embryo by single-cell transcriptomics combined with spatial mapping.

    (A) Single-cell sequencing of the Drosophila embryo: ~1000 handpicked stage 6 fly embryos are dissociated per Drop-seq replicate, cells are fixed and counted, single cells are combined with barcoded capture beads, and libraries are prepared and sequenced. Finally, single-cell transcriptomes are deconvolved, resulting in a digital gene expression matrix for further analysis. (B) Mapping cells back to the embryo: Single-cell transcriptomes are correlated with high-resolution gene expression patterns across 84 marker genes, cells are mapped to positions within a virtual embryo, and expression patterns are computed by combining the mapping probabilities with the expression levels (virtual in situ hybridization).

  • Fig. 2 Reconstructing the embryo by spatial mapping based on distributed scores.

    (A) DistMap. The 84 BDTNP gene expression patterns (I) and the single-cell expression profiles (II) were binarized. (III) Confusion matrices are calculated scoring expression (dis)agreement between the transcriptomes and the ~3000 positional bins of the reference atlas. Matthews correlation coefficients (MCCs) are calculated for every cell/bin combination. (IV) Positional assignment for each cell is distributed based on MCCs across all bins. (B) Density plot showing mapping confidence (mean Euclidean distance) between a cell’s highest scoring location and the following six locations. Single-cell transcriptomes (red) map to embryo positions with similar confidence as cells of the reference atlas (green). (C) Bin coverage across the embryo. More than 87% of all locations in the embryo are confidently covered (P < 0.05; see the supplementary materials for details). (D) The virtual fly embryo has a resolution of 6000 to 8000 genes per cell.

  • Fig. 3 Sequenced cells cluster by spatial identity.

    (A) Two-dimensional t-SNE representation of the high-quality cells shows nine major clusters grouped by transcriptome similarity. (B) Mapping of clusters reveals that cells within each cluster share a contiguous spatial domain.

  • Fig. 4 DVEX accurately predicts spatial gene expression patterns.

    DVEX is the online resource for the virtual embryo. (A) Virtual in situ hybridization (vISH) for the pair rule gene ftz (red) and the mesodermal gene sna (green) in five orientations. Stippled box indicates cells analyzed in (B). EL, egg length; DV, dorsoventral; AP, anteroposterior. (B) Quantification of relative expression per cell mapped along an axis (here, dorsoventral) for stumps (expressed in the ventral mesoderm, left) and the vnd::DsRed reporter (primarily expressed in the ventral neurectoderm, right). Relative expression in log space; thresholds were 0.85, embryos are oriented anterior left. (C) Examples of marker genes and their expression in t-SNE clustered cells. Expression indicated, gray (low) to red (high).

  • Fig. 5 Prediction accuracy and detection of new regulators.

    (A) vISH predictions are accurate across a wide variety of expression patterns. Expression of CGs had not been reported previously. (B) Patterned expression of putative transcription factors. (C) Patterned expression of lncRNAs. (D) CR43432 and pan-neurogenic genes are expressed in complementary patterns. Dual vISH of SoxN and CR43432 (top left); double in situ hybridization validates the predicted expression. CR43432 is additionally expressed in yolk nuclei (not shown in vISH).

  • Fig. 6 Spatial regulation of Hippo signaling in the embryo.

    (A) vISHs predict patterned expression of Hippo signaling components in stage 6 embryos. Shown are components involved in receiving the signal (receptors and ligands), transducing it through the cytoplasm (transducers) and inhibition of the transcriptional cofactor Yorkie (Yki). Ubiquitous pathway components are indicated; vISHs for patterned components are shown. Most patterned positive Hippo components are expressed in anterior regions. Active signaling culminates in nuclear exclusion of Yki. (B) Shown is the anterior of a stage 7 embryo; cephalic furrow is indicated by stippled white line, anterior left. Staining with antibodies against phosphorylated histone H3 (H3S10-P) marks cells undergoing mitosis; nuclear Yki is depleted in cells not marked by H3S10-P.

Supplementary Materials

  • The Drosophila embryo at single-cell transcriptome resolution

    Nikos Karaiskos, Philipp Wahle, Jonathan Alles, Anastasiya Boltengagen, Salah Ayoub, Claudia Kipar, Christine Kocks, Nikolaus Rajewsky, Robert P. Zinzen

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

    Download Supplement
    • Materials and Methods
    • Supplementary Text
    • Figs. S1 to S8
    • Tables S1 to S5
    • References
    Table S6
    Pole cell marker genes
    Table S7
    BDTNP reference atlas – thresholds and expression matrix
    Table S8
    Homology analysis

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