FIGURE SUMMARY
Title

scMultiome analysis identifies embryonic hindbrain progenitors with mixed rhombomere identities

Authors
Kim, Y.I., O'Rourke, R., Sagerström, C.G.
Source
Full text @ Elife
Figure 1—figure supplement 1.

(A). UMAP of 13hpf neural clusters. (B). Dot plot showing the expression of the top five enriched genes in each cluster. (C, D). Feature plots showing the expressing of sox3 (C) and neurod4 (D). (E-L). Expression of rhombomere-specific genes are shown as feature plots (left panels) and violin plots (right panels). (M-P). Feature plots showing the expression of dorsoventral marker genes. (Q-T). Hybridization chain reaction (HCR) analysis of rhombomere-restricted gene expression in 13hpf wild-type zebrafish embryos. Embryos are shown in dorsal view with anterior to the top. FB = forebrain, MB = midbrain, MHB = midbrain-hindbrain boundary, r=rhombomere, CHB = caudal hindbrain, SC = spinal cord. Scale bar in (Q-T) = 100 um.

Figure 1—figure supplement 1.

(A). UMAP showing all clusters obtained from 13hpf dissected zebrafish hindbrain regions. (B). Heatmap showing the expression of the top five enriched genes in each cluster. (C). Dot plot showing the expression of the top five enriched genes in each cluster.

Figure 2—figure supplement 1.

(A) UMAP of 16hpf neural clusters. (B) Dot plot showing the expression of the top five enriched genes in each cluster. (C, D) Feature plots showing the expressing of sox3 (C) and neurod4 (D). (E, F) Feature plots showing the expression of dorsoventral marker genes. dorsNT = dorsal neural tube, NC = neural crest. See legend to Figure 1 for additional abbreviations.

Figure 2—figure supplement 1.

scMultiome analysis of 16hpf zebrafish.

(A). UMAP showing all clusters obtained from 16hpf dissected zebrafish hindbrain regions. (B). Heatmap showing the expression of the top five enriched genes in each cluster. (C). Dot plot showing the expression of the top five enriched genes in each cluster.
Figure 3—figure supplement 1.

(A) UMAP of 10hpf neural clusters. (B, C) Heat map (B) and dot plot (C) showing the expression of the top five enriched genes in each cluster. (D-K) Expression of rhombomere-specific genes are shown as feature plots (left panels) and violin plots (right panels). (L-M) Feature plots showing the expressing of sox3 (L) and neurod4 (M). (N, O) Feature plots showing the expression of dorsoventral marker genes. (P) Dendrogram showing the relationship between the 10hpf neural clusters. (Q-V) Rhombomere-enriched accessible transcription factor binding motifs are shown as a motif logo (left panels) and as a feature plot of chromVar activity (right panel). HB = hindbrain, NC = neural crest. See legend to Figure 1 for additional abbreviations.

Figure 3—figure supplement 1.

scMultiome analysis of 10hpf zebrafish.

(A) UMAP showing all clusters obtained from 10hpf whole zebrafish embryos. (B) Heatmap showing the expression of the top five enriched genes in each cluster. (C) Dot plot showing the expression of the top five enriched genes in each cluster.
Figure 4—source data 1.

Transcription factor binding motifs show rhombomere-restricted accessibility.

See also Figure 4—source data 1. (A, B). Feature plots showing the expression of TEAD transcription factors at 13hpf and 16hpf. (C-J). Rhombomere-enriched accessible transcription factor motifs are shown as a motif logo (left panels) and as a feature plot of chromVar activity at 13hpf (middle panels), or 16hpf (right panels).

Differentially accessible transcription factor motifs for each cluster at each time point.

Figure 5—figure supplement 1.

Each rhombomere displays a unique molecular profile. See also Figure 5—figure supplements 1–7. (A, B) Heat maps showing the expression of the top five enriched genes in each cluster at 13hpf (A) and 16hpf (B). Gene order in A and B is the same as in Figures 1B and 2B, respectively. (C, D). Dendrograms showing the relationship between each neural cluster at 13hpf (C) and 16hpf (D). ( E-I). Violin plots showing expression levels of genes enriched in odd (E) or even (F) rhombomeres, as well as genes enriched in adjacent pairs of rhombomeres (G–I), at 13hpf.

Figure 5—figure supplement 1.

Direct-Net derived gene regulatory network for rhombomere 1 at 13hpf. TFs (squares) and non-TFs (ovals) are linked by the presence of accessible TF motifs within 500 bp (solid lines) or 250 kb (dashed lines) of a gene’s transcription start site.

Figure 5—figure supplement 2.

Direct-Net derived gene regulatory network for rhombomere 2 at 13hpf.

TFs (squares) and non-TFs (ovals) are linked by the presence of accessible TF motifs within 500 bp (solid lines) or 250 kb (dashed lines) of a gene’s transcription start site.
Figure 5—figure supplement 3.

Direct-Net derived gene regulatory network for rhombomere 3 at 13hpf.

TFs (squares) and non-TFs (ovals) are linked by the presence of accessible TF motifs within 500 bp (solid lines) or 250 kb (dashed lines) of a gene’s transcription start site.
Figure 5—figure supplement 4.

Direct-Net derived gene regulatory network for rhombomere 4 at 13hpf.

TFs (squares) and non-TFs (ovals) are linked by the presence of accessible TF motifs within 500 bp (solid lines) or 250 kb (dashed lines) of a gene’s transcription start site.
Figure 5—figure supplement 5.

Direct-Net derived gene regulatory network for rhombomere 5 at 13hpf.

TFs (squares) and non-TFs (ovals) are linked by the presence of accessible TF motifs within 500 bp (solid lines) or 250 kb (dashed lines) of a gene’s transcription start site. Due to the large network produced for r5, only nodes with more than eight connections are shown.
Figure 5—figure supplement 6.

Direct-Net derived gene regulatory network for rhombomere 6 at 13hpf.

TFs (squares) and non-TFs (ovals) are linked by the presence of accessible TF motifs within 500 bp (solid lines) or 250 kb (dashed lines) of a gene’s transcription start site.
Figure 5—figure supplement 7.

Direct-Net derived Egr2 networks for rhombomeres 3 and 5 at 13hpf.

The Egr2 networks for r3 and r5 were computationally combined, and color-coded such that Egr2 nodes observed only in r3 are indicated in yellow, ones observed only in r5 are blue, and Egr2 nodes shared between r3 and r5 are green. TFs (squares) and non-TFs (ovals) are linked by the presence of accessible TF motifs within 500 bp (solid lines) or 250 kb (dashed lines) of a gene’s transcription start site.
Figure 6—figure supplement 1.

Characterization of gene expression in primary hindbrain progenitor domains (PHPDs) relative to their derivative rhombomeres.

(A). Dendrogram showing the relationship between the integrated neural clusters. (B). Co-expression of r2 markers (vgll3) and r3 markers (egr2b, nab1b) in individual cells of HB.1. Numbers in the bar graph represent cell counts. (C). Heatmap showing genes differentially expressed between HB.1 at 10hpf and its derivative rhombomeres (r2 and r3) at 13hpf. (D-H). Feature plots showing the gene expression in HB.2 at 10hpf relative to r5/r6 at 13hpf.
Figure 6—figure supplement 1.

Characterization of gene expression in primary hindbrain progenitor domains (PHPDs) relative to their derivative rhombomeres.

(A). Dendrogram showing the relationship between the integrated neural clusters. (B). Co-expression of r2 markers (vgll3) and r3 markers (egr2b, nab1b) in individual cells of HB.1. Numbers in the bar graph represent cell counts. (C). Heatmap showing genes differentially expressed between HB.1 at 10hpf and its derivative rhombomeres (r2 and r3) at 13hpf. (D-H). Feature plots showing the gene expression in HB.2 at 10hpf relative to r5/r6 at 13hpf.
Figure 7—figure supplement 1.

scMultiome analysis of DEAB-treated 13hpf zebrafish.

(A). UMAP showing all clusters obtained from 13hpf dissected zebrafish hindbrain regions treated with DEAB. (B). Heatmap showing the expression of the top five enriched genes in each cluster. (C). Dot plot showing the expression of the top five enriched genes in each cluster.
Figure 7—figure supplement 1.

scMultiome analysis of DEAB-treated 13hpf zebrafish.

(A). UMAP showing all clusters obtained from 13hpf dissected zebrafish hindbrain regions treated with DEAB. (B). Heatmap showing the expression of the top five enriched genes in each cluster. (C). Dot plot showing the expression of the top five enriched genes in each cluster.
Figure 7—figure supplement 2.

DEAB treatment has a limited effect on gene expression in rhombomeres 1–4.

(A-E). Volcano plots showing differential gene expression between control and DEAB-treated cells for each rhombomere. A positive log2FC indicates higher expression in control than DEAB.
Figure 8.

Proposed model for the transition from mixed identity primary hindbrain progenitor domain (PHPD) cells to rhombomere cells.

An early ground state that lacks an anteroposterior pattern is proposed to respond to morphogens (fibroblast growth factor (Fgf) anteriorly and retinoic acid posteriorly) to generate three PHPDs. Each PHPD contains progenitor cells with mixed identities (HB.1=r1, r2, r3; HB.2=r5, r6, HB.3=r4) that are subsequently resolved via repressive TF interactions (red lines) into rhombomere cells with a single transcriptional identity.
Acknowledgments
This image is the copyrighted work of the attributed author or publisher, and ZFIN has permission only to display this image to its users. Additional permissions should be obtained from the applicable author or publisher of the image. Full text @ Elife
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