Heterogeneous Expression of tcf21, tbx18, and wt1b in the Developing Zebrafish Epicardium

(A) Fluorescence of tcf21:myr-tdTomato (magenta membrane), tbx18:myr-eGFP (green membrane), and wt1b:H2B-Dendra2 (green nucleus).

(B) Workflow to establish TgBAC(tcf21:myr-tdTomato;tbx18:myr-eGFP;wt1b:H2B-Dendra2)^ox187. (C) Schematic of epicardial fluorescence patterns and cardiac regions analyzed. dsBA, distal bulbus arteriosus; prBA, proximal bulbus arteriosus; apV, arterial pole of the ventricle; and AVC, atrioventricular canal.

(D–F) Single optical sections of mRNA stainings of (D) tcf21 (cyan) and tcf21:myr-tdTomato (magenta), (E) tbx18 (magenta) and tbx18:myr-eGFP (green), and (F) wt1b (magenta) and wt1b:H2B-Dendra2 (green). (D′–F′) Cell nuclei (asterisks) in the epicardial region surrounded by endogenous mRNA and fluorophore mRNA/protein.

(G–I) Projections of the heart in triple reporter larvae at 3 dpf (G), 5 dpf (H), and 7 dpf (I).

(G′ and H′) Single optical sections from (G) and (H).

(I′) Short projection from (I).

(G″–I″ and G‴–I‴) Representative epicardial fluorescence patterns.

(J–L) Relative quantification of epicardial fluorescence patterns across the regions indicated in (C) at 3 dpf (J), 5 dpf (K), and 7 dpf (L).

Scale bars: 20 μm in (D)–(F), 5 μm in (D′)–(F′), 50 μm in (G)–(I) and (G′)–(I′), and 10 μm in (G″)–(I″) and (G‴)–(I‴). Color channels were adjusted separately for brightness and contrast. Data in (J)–(L) are represented as mean minus standard deviation. Number of embryos analyzed: 3 dpf n = 6, 5 dpf n = 10, and 7 dpf n = 6. V, ventricle; BA, bulbus arteriosus.

See also Figure S1.

Single-Cell RNA Sequencing Reveals Distinct Epicardial Cell Clusters in the Developing Zebrafish Heart

(A) Overview of the scRNA-seq workflow.

(B) t-distributed stochastic neighbor embedding (t-SNE) clustering of single-cell samples. Colors indicate the reporter or WT line cells were isolated from.

(C) Identification of single-cell clusters based on marker gene expression. Three clusters labeled by tcf21, tbx18, and wt1b are designated Epi1-3. Color key indicates range of log₂ transformed fragments per kilobase of transcript per million reads mapped (FPKM) expression values.

(D) GO term over-representation in the clusters (columns). Bubble size depicts fold enrichment and color scale depicts the statistical significance, with p-values calculated using Fisher’s exact test and Bonferroni correction for multiple hypothesis testing. GO terms important for further analysis are highlighted in red.

(E) Relative quantification of the combinations of tcf21, tbx18, and wt1b expression in epi1–3 cells. CM, cardiomyocyte; eHC, erythroid hematopoietic cell; mHC, myeloid hematopoietic cell; NC, neural cell; and MC, mesenchymal cell.

See also Figures S2 and S3.

Transcriptionally Distinct Epicardial Subpopulations Epi1–3 Localize to Different Regions of the Developing Heart

(A) Marker gene expression in Epi1–3. Cells were clustered in an unsupervised manner (columns). Color key indicates range of log₂ transformed FPKM values. Genes analyzed further are highlighted in red. CM, cardiomyocyte; eHC, erythroid hematopoietic cell; mHC, myeloid hematopoietic cell; NC, neural cell; and MC, mesenchymal cell.

(B) mRNA staining of the Epi1 markers adma (magenta), jam2b (red), and tcf21 (cyan) in a 5 dpf heart. (B′ and B″) A nucleus (asterisk) in the epicardial region surrounded by adma, jam2b, and tcf21.

(C) mRNA staining of the Epi2 marker elnb (magenta) and tbx18 (green) at 5 dpf. (C′ and C″) Overlap of elnb and tbx18 in the BA.

(D) Antibody staining of Mylka and tbx18:myr-Citrine at 5 dpf. (D′ and D″) Overlap of Mylka and myr-Citrine in the BA.

(E) mRNA staining of the Epi3 marker cldn11a (magenta) and tcf21 (cyan) at 5 dpf. (E′ and E″) Two nuclei (asterisks) in the epicardial region between BA and atrium in close proximity to cldn11a and tcf21. Scale bars: 20 μm in (B)–(E) and 5 μm in (B′)–(E′) and (B″)–(E″). Color channels were adjusted separately for brightness and contrast. (B)–(E) are single optical sections. V, ventricle; BA, bulbus arteriosus.

Transglutaminase 2b Is Enriched in Epi1 and Functions to Maintain the Integrity of the Epicardial Cell Layer

(A) Expression of tgm2b. Color key indicates range of log₂ transformed FPKM values.

(B) mRNA staining of tgm2b (magenta), tcf21 (cyan), and tbx18 (orange) in a 5 dpf heart. (B′ and B″) A nucleus (asterisk) in the epicardial region surrounded by tgm2b, tcf21, and tbx18.

(C) The epicardium in a 5 dpf control larva.

(D) Disrupted epicardial integrity in a 5 dpf KO tgm2b larva.

(E) Absolute quantification of tcf21:H2B-Dendra2⁺ epicardial cell numbers at 5 dpf in transient tgm2b knockouts (Control, KO tgm2b) and stable tgm2b mutants (heterozygous, tgm2b^wt/mut; homozygous, tgm2b^mut/mut). Larvae from two mutant lines were analyzed.

(F) Absolute quantification of tbx18:myr-Citrine⁺ epicardial cell numbers at 5 dpf.

(G) The epicardium in a 66 hpf control embryo. White arrows indicate epicardial cells, and red arrowheads indicate cells in the PEO.

(H) Reduced epicardial cell numbers in a 66 hpf KO tgm2b embryo.

(I and J) Absolute quantification of tcf21:H2B-Dendra2⁺ epicardial (I) and proepicardial (J) cell numbers at 66 hpf.

(K) Proliferation in the epicardium of a 77 hpf control larva (white arrows).

(L) Increased epicardial proliferation in a KO tgm2b larva.

(M) Relative quantification of proliferating epicardial cells.

(N) Absolute quantification of tcf21:H2B-Dendra2⁺ epicardial cells. Scale bars: 50 μm in (C), (D), (G), and (H); 20 μm in (B); 10 μm in (K) and (L); and 5 μm in (B′) and (B″). Color channels were adjusted separately for brightness and contrast. (C), (D), (G), and (H) are projections; (B), (K), and (L) are single optical sections. Data are represented as median, first, and third quartiles (box). Significance was calculated using Welch’s t test. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001. V, ventricle; Ab, antibody.

See also Figure S6.

Semaphorin 3fb Is a Marker of Epi2 and Controls the Number of tbx18⁺ Cells in the Bulbus Arteriosus

(A–C) Expression of sema3fb (A), nrp1a (B), and nrp2a (C). Color key indicates range of log₂ transformed FPKM values.

(D) mRNA staining of sema3fb (cyan), nrp2a (red, arrowheads), nrp1a (magenta, arrows), and tbx18 (orange) at 5 dpf. (D′) Close proximity of nrp2a, nrp1a, and tbx18 to a nucleus (asterisk) at the BA boundary.

(E) mRNA staining of nrp2a (red), nrp1a (magenta), and tcf21 (cyan) at 5 dpf. (E′) A nucleus (asterisk) in close proximity to nrp2a, nrp1a, and tcf21.

(F) The BA in a 5 dpf control larva.

(G) Increased numbers of tbx18:myr-Citrine⁺ cells (arrows) in the BA of a KO sema3fb larva.

(H) Absolute quantification of tbx18:myr-Citrine⁺ cell numbers in the BA at 5 dpf.

(I–N) Analysis of gene editing in single sema3fb-mutated epicardial cells. (I) t-SNE plot showing cell clusters and editing events. Numbers indicate cell identities, and colors depict editing events. Ld, large deletion; fs, frameshift; and sub, substitution. (J) Relative number of Cas9-edited sema3fb⁺ cells. Colors indicate the sema3fb sgRNA target site edited. (K) Frequency of editing event types. (L) Inferred edited sema3fb protein lengths, relative to WT. (M) Sashimi plots showing splicing of sema3fb. Arrows highlight reads splicing abnormally. Red bars indicate sgRNA target sites. (N) Relative number of sema3fb splicing reads that spliced abnormally.

Scale bars: 10 μm in (D)–(G) and 5 μm in (D′) and (E′). Color channels were adjusted separately for brightness and contrast. (D)–(G) are single optical sections. Data in (H) and (L) are represented as median, first, and third quartiles (box). Significance calculated using Welch’s t test. ^∗∗p < 0.01. V, ventricle; BA, bulbus arteriosus.

See also Figure S6.

The Chemokine cxcl12a Is Expressed in Epi3 and Attracts ptprc⁺ Leukocytes to the Epicardium

(A) Expression of cxcl12a. Color key indicates range of log₂ transformed FPKM values.

(B) mRNA staining of cxcl12a (magenta) and tcf21 (cyan) at 5 dpf. (B′ and B″) A nucleus (asterisk) in the epicardial region between BA and atrium surrounded by cxcl12a and tcf21.

(C and D) Expression of cxcr4b (C) and ptprc (D). Color key indicates range of log₂ transformed FPKM values.

(E) mRNA staining of cxcr4b (green), ptprc (magenta), and tcf21 (cyan) at 5 dpf. (E′) A nucleus (asterisk) in close proximity to cxcr4b and ptprc.

(F) The heart in a 5 dpf control larva. (F′ and F″) ptprc/Cd45:DsRed⁺ cells in contact with the epicardium (arrows).

(G and G′) Fewer ptprc/CD45:DsRed⁺ cells on the heart of a 5 dpf KO cxcl12a larva.

(H) Absolute quantification of ptprc/CD45:DsRed⁺ cells in contact with the epicardium in transient cxcl12a knockouts (Control, KO cxcl12a) and stable cxcl12a mutants (heterozygous, cxcl12a^wt/mut; homozygous, cxcl12a^mut/mut).

(I and J) RNA-seq analysis of cxcl12a gene editing in single control (ctr) and cxcl12a-knockout (KO) larvae. Relative numbers of edited reads that spanned the target site of cxcl12a sgRNA1 (I) or sgRNA3 (J).

(K) Sashimi plots showing splicing of cxcl12a. Arrows highlight reads splicing from exon 1 into exon 3. Red bars indicate sgRNA target sites.

(L) Relative number of reads splicing from cxcl12a exon 1 that spliced from exon 1 into exon 3.

Scale bars: 50 μm in (F) and (G); 20 μm in (B); 10 μm in (F′), (F″), and (G′), and 5 μm in (B′), (B″), (E), and (E′). Color channels were adjusted separately for brightness and contrast. (F) and (G) are projections; (B), (E), (F′), (F″), and (G′) are single optical sections. Data in (H) and (L) are represented as median, first, and third quartiles (box). Significance calculated using Welch’s t test. ^∗∗p < 0.01, ^∗∗∗p < 0.001. V, ventricle; eHC, erythroid hematopoietic cell; mHC, myeloid hematopoietic cell.

See also Figure S7.

Summary of the Distinct Epicardial Cell Populations in the Developing Zebrafish Heart

(A) Zebrafish heart at 5 dpf, showing the distributions of Epi1-3.

(B) Loss of tgm2b disrupts the integrity of the epicardial cell layer.

(C) Loss of sema3fb increases the number of Epi2 cells in the BA, possibly owing to increased cell migration from the epicardium.

(D) Loss of cxcl12a decreases the number of myeloid cells on the heart. V, ventricle; BA, bulbus arteriosus.