Research ArticleINNATE LYMPHOID CELLS

Single-cell transcriptional analysis reveals ILC-like cells in zebrafish

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Science Immunology  16 Nov 2018:
Vol. 3, Issue 29, eaau5265
DOI: 10.1126/sciimmunol.aau5265
  • Fig. 1 Rag1−/− zebrafish have cytokine-producing cells in the gut.

    (A) Representative FACS plots showing the percentage of cells in the lymphocytes’ gate (as defined by FSC/SSC gating) in the gut of wild-type zebrafish (left) and rag1−/−mutant (right). (B) Percentage of cells in the lymphocytes’ gate within 50,000 recorded events in the gut of wild-type and rag1−/−mutant zebrafish. Bars represent the geometric mean ± 95% confidence interval to estimate the total number of lymphocytes. Mann-Whitney test. (C) qPCR expression of T cell–associated markers (cd3z and trac) and lymphocytes’ markers (il7r and lck) in mutant and wild-type zebrafish. Bars represent the geometric mean ± 95% confidence interval to estimate fold changes. Mann-Whitney test. (D) Scheme of short-term inflammation experiment. IP, intraperitoneal. (E) qPCR expression of immune type 1 (ifng1-1 and ifng1-2), immune type 2 (il4 and il13), and immune type 3 (il17a/f3 and il22) signature cytokines in the gut of the wild-type (rag1+/+) and mutant (rag1−/−) zebrafish after 6 hours of immune challenge with V. anguillarum or A. simplex. Bars represent the geometric mean ± 95% confidence interval to estimate fold changes. One-way analysis of variance (ANOVA) test.

  • Fig. 2 Analysis of the lck+ cells, collected from the gut of rag1−/− zebrafish.

    (A) PBS-injected zebrafish. (B) V. anguillarum–injected zebrafish. (C) A. simplex–injected zebrafish. The 2D projection of tSNE analysis of 10x RNA-seq data showing heterogeneity of ILCs. Dot plots show the level of expression of marker genes and the percentage of cells per cluster that express the gene of interest.

  • Fig. 3 Analysis of the lck+ cells collected from the gut of wild-type zebrafish.

    (A) The 2D projection of tSNE analysis of 10x RNA-seq data showing heterogeneity of innate and adaptive lymphocytes’ pool. (B) Dot plot shows the level of expression of signature genes and the percentage of cells per cluster that express the gene of interest.

  • Fig. 4 Integrated analysis of PBS-, V. anguillarum–, and A. simplex–injected rag1−/− zebrafish.

    (A) Dot plot with the expression level of selected marker genes in each of the clusters. The size of the dots indicates the percentage of cells within the cluster that express the gene of interest; each cluster contains cells from three different conditions. (B) Volcano plot showing the top 20 differentially expressed genes between nitr+rorc+ (cluster 7) and nitrrorc+ (cluster 13) cells originated from rag1−/− PBS-injected zebrafish using aligned dataset. FC, fold change.

  • Fig. 5 Genotype clustering of cells from the rag1−/− zebrafish.

    (A) PBS-injected zebrafish. (B) V. anguillarum–injected zebrafish. (C) A. simplex–injected zebrafish. The first three PCs were used, and the clusters were generated using the probabilistic PCA algorithm. Individual 1 (violet color), individual 2 (green color), individual 3 (pink color), and undetermined (light violet color). Bar plots showing frequency (log scaled) of different donor within the cell type clusters under different challenge conditions.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/3/29/eaau5265/DC1

    Table S1. TraCeR and BraCeR analysis.

    Table S2. Number of cells in each cluster across different experimental conditions (10x RNA-seq data).

    Table S3. Index sorting data.

    Fig. S1. Expression of marker genes in human ILCs.

    Fig. S2. QC of 10x data.

    Fig. S3. Heatmaps with expression of marker genes in lck+ cells collected from rag1−/− gut.

    Fig. S4. t-SNE plots with expression of marker genes in lck+ cells collected from rag1−/− gut.

    Fig. S5. Identification of nitrrorc+ and nitr+rorc+ population of cells.

    Fig. S6. PBS-, V. anguillarum–, and A. simplex–injected rag1−/− mutant zebrafish aligned datasets.

    Fig. S7. Identification of immune cell types in zebrafish during steady-state hematopoiesis.

    Fig. S8. QC of SmartSeq2 data.

    Fig. S9. Clustering of Smart-seq2 data from Tg(lck:EGFP), Tg(cd4-1:mCherry), and Tg(mhc2dab:GFP, cd45:dsRed) zebrafish transgenic lines.

  • Supplementary Materials

    The PDF file includes:

    • Legends for table S1 and S3
    • Table S2. Number of cells in each cluster across different experimental conditions (10x RNA-seq data).
    • Fig. S1. Expression of marker genes in human ILCs.
    • Fig. S2. QC of 10x data.
    • Fig. S3. Heatmaps with expression of marker genes in lck+ cells collected from rag1−/− gut.
    • Fig. S4. t-SNE plots with expression of marker genes in lck+ cells collected from rag1−/− gut.
    • Fig. S5. Identification of nitrrorc+ and nitr+rorc+ population of cells.
    • Fig. S6. PBS-, V. anguillarum–, and A. simplex–injected rag1−/− mutant zebrafish aligned datasets.
    • Fig. S7. Identification of immune cell types in zebrafish during steady-state hematopoiesis.
    • Fig. S8. QC of SmartSeq2 data.
    • Fig. S9. Clustering of Smart-seq2 data from Tg(lck:EGFP), Tg(cd4-1:mCherry), and Tg(mhc2dab:GFP, cd45:dsRed) zebrafish transgenic lines.

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    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). TraCeR and BraCeR analysis.
    • Table S3 (Microsoft Excel format). Index sorting data.

    Files in this Data Supplement:

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