Chestnut et al., 2020 - Single-cell transcriptomic analysis identifies the conversion of zebrafish Etv2-deficient vascular progenitors into skeletal muscle. Nature communications   11:2796 Full text @ Nat. Commun.

Fig. 1 Single-cell RNA-seq analysis of <italic>etv2</italic><sup><italic>ci32Gt</italic></sup><italic>; UAS:GFP</italic> heterozygous and homozygous embryos at the 20-somite stage.

a, b t-SNE plots and cell clustering analysis. Twelve different clusters were identified. Card cardiomyocytes, EC endothelial cells, EPC endothelial progenitor cells, Epid epidermal cells, LPM lateral plate mesoderm, Macr macrophages, Myoc myocytes, Neur neural, Apo putative pro-apoptotic cells, RBC red blood cells, Tailbud tailbud progenitors. c t-SNE plots showing selected top markers for different cell types. d Heatmap of marker gene expression in different cell populations. e The proportions of GFP+ cell types in etv2ci32Gt+/− and etv2ci32Gt−/− embryos. Note a great reduction in EC1, EC2, EPC, and macrophage populations and an increase in LPM, cardiomyocyte, myocyte, RBC, and Apo populations in etv2 mutants. ***p < 0.001, NS not significant, chi-square test. p Values: Card—5.0 × 10−26, EC1— 6.2 × 10-18, EC2—1.8 × 1014, EPCs—2.4 × 10−11, Epid—0.66, LPM—8.4 × 10−27, Macr—2.5 × 10−16, Myoc—3.4 × 10−4, Neur—1.0, Apo—1.9 × 10−7, RBC—1.1 × 10−4, Tailbud—0.21. Totally, 2049 and 588 cells total from etv2ci32Gt+/− and etv2ci32Gt−/− embryos, respectively, were analyzed in a single scRNA-seq experiment. f Pseudotime analysis graph of cells in LPM, EPC, EC1, EC2, and myocyte populations in etv2ci32Gt homozygous and heterozygous embryos.

Fig. 2 Identification of the transcriptional signature of endocardial progenitors.

Subclustering of the EC1 population in etv2ci32Gt+/− embryos identified a novel endocardial subpopulation (while the remaining endothelial cells are designated as EC1a). a A section of a heatmap showing marker gene expression in endocardial, endothelial (EC1a and EC2), endothelial progenitor (EPC), and cardiomyocyte populations. b A portion of the global t-SNE plot showing the endocardial subpopulation. c Heatmap for subclustering of EC1 population showing genes enriched in endocardial and the remaining endothelial (EC1a) cells. d, e t-SNE and violin plots for selected endocardial genes. fi In situ hybridization analysis at the 20-somite stage for selected endocardial-enriched genes fn1a and drl (f, g arrows point to endocardial expression) and endocardial-excluded genes etv2 and lmo2 (bilateral expression is present in cranial endothelial progenitors but is largely absent from the endocardial cluster). Flat-mounted embryos, ventral view, anterior is to the left. The number of embryos displaying the representative phenotype out of the total number of embryos obtained from two replicate experiments is shown.

Anatomical Term:
Stage: 20-25 somites

Fig. 3 <italic>Etv2-</italic>expressing cells differentiate as skeletal muscle cells in the absence of <italic>etv2</italic> function.

ad Trunk region of etv2ci32Gt; UAS:GFP embryos at 25 hpf. Maximum intensity projections of selected confocal slices are shown in (c, d). Note the absence of intersegmental vessels (ISVs) and elongated GFP-positive skeletal muscle fibers (arrows, c, d). The embryos were obtained from an incross of heterozygous etv2ci32Gt carriers; embryo numbers (lower right) correspond to the expected Mendelian ratio. e Quantification of GFP+ myocytes in the trunk region of 8 etv2ci32Gt+/− and 10 etv2ci32Gt−/−; UAS:GFP embryos at 25 hpf, analyzed in 2 replicate experiments. The bars show median values. fh Co-expression of etv2ci32Gt+/−; UAS:NTR-mCherry and muscle-specific actc1b:GFP in mCherry-positive muscle cells (arrows, g, h). ik The Tg(etv2:mCherry) line shows mCherry expression in skeletal muscle cells when crossed to etv2ci32Gt+/−; UAS:GFP carriers. l, m Multiple GFP+ skeletal muscle cells are apparent in the progeny of etv2ci32Gt+/−; UAS:GFP zebrafish crossed with the etv2ci33+/− line, which carries a loss-of-function mutation in etv2. Note that the expected frequency of double heterozygous embryos in (m) is 50%. n Quantification of GFP+ myocytes in the trunk region of etv2ci32Gt/ci33 and etv2ci32Gt+/−; scl MO embryos shown in (m, p) obtained in two replicate experiments. The graphs show median and SD values. o GFP+ skeletal muscle cells observed in etv2y11−/− embryos crossed into the Tg(-2.3 etv2:GFP) reporter line. p Multiple GFP + skeletal muscle cells are apparent in etv2ci32Gt+/−; UAS:GFP embryos injected with scl MO. See graph n for quantification. qs Ectopic myocytes observed in scl MO-injected etv2ci32Gt+/−; UAS:NTR-mCherry embryos are positive for muscle-specific actc1b:GFP expression at 24 hpf. tetv2 RNA overexpression inhibits myod expression (arrow). Dorsal view, anterior is to the left. u qPCR analysis of myf5, myod, and myog expression in etv2 RNA-injected and uninjected control embryos at the 10-somite stage. Mean values ± SEM is shown. RNA was purified from groups of ten embryos analyzed in two replicate experiments. In all graphs, two-tailed Student’s t test was used. The number of embryos displaying the representative phenotype out of the total number of embryos obtained from two replicate experiments is shown.

Fig. 4 Time-lapse imaging of cell migration in <italic>etv2</italic><sup><italic>ci32Gt</italic></sup> embryos starting at the 9–10-somite stage.

Lateral view is shown, anterior is to the left. af In etv2ci32Gt+/−; UAS:GFP embryos, bilaterally located vascular and hematopoietic progenitors within the lateral plate mesoderm (LPM, arrowheads) migrate toward the midline and coalesce into the axial vasculature (arrows). Note that some cells remain in the lateral position and elongate into muscle cells (myoc). DA progenitors of the dorsal aorta, ISV intersegmental vessels. Time frames are selected from the Supplementary Movie 1. gl In etv2ci32Gt−/−; UAS:GFP embryos, cells initiate migration (blue arrows) but fail to coalesce into the axial vasculature. Instead, many cells either undergo apoptosis (apo, red arrowheads point to round apoptotic cells) or differentiate into myocytes (white arrows, myoc). Time frames are selected from the Supplementary Movie 3. m Higher magnification view showing differentiation of a GFP+ progenitor cell initially positioned in the LPM into a myocyte (arrowhead points to the same cell). Note that the cell migrates dorsally from the LPM into the somite and then elongates as it undergoes differentiate into the muscle. Time frames are selected from the Supplementary Movie 4. Representative embryos are shown out of the total of seven heterozygous and four homozygous etv2ci32Gt; UAS:GFP embryos that were imaged in two replicate experiments.

Fig. 5 Fluorescent in situ hybridization for <italic>myod</italic> expression in <italic>etv2</italic><sup><italic>ci32Gt+/−</italic></sup><italic>; UAS:GFP</italic> embryos.

In situ hybridization was performed using hybridization chain reacion (HCR) at the 8–10-somite stages. ac Maximum intensity projection is shown; df An area boxed in a was imaged at higher magnification; maximum intensity projection of three confocal slices is shown. Note that most GFP-expressing cells are positioned in the lateral plate mesoderm, while some cells are starting to migrate toward the midline (arrow, d, e, points to a migrating angioblast which does not have myod expression). GFP and myod co-expressing cells (arrowheads, d, e) are apparent at the posterior edge of the somite. The punctate myod expression pattern is due to the nature of the HCR probe. Dorsolateral view, anterior is to the left. The numbers in the lower left corner display the number of embryos showing the phenotype out of the total number of embryos analyzed in two replicate experiments.

Fig. 6 Wnt and FGF signaling is required for differentiation of <italic>etv2</italic><sup><italic>ci32Gt</italic></sup><italic>; UAS:GFP</italic> cells into skeletal muscle.

ac Heat-shock inducible expression of Wnt inhibitor Dkk1 greatly reduces GFP-positive muscle cells in hsp70:dkk1; etv2ci32Gt+/−; UAS:GFP embryos. Heat-shock (HS) was performed at the 8-somite stage, and GFP fluorescence imaged at 24–26 hpf. Sixteen embryos were analyzed in each group in two replicate experiments. dfetv2ci32Gt−/−; UAS:GFP embryos treated with FGF inhibitor SU5402 starting at the 50% epiboly stage show a greatly reduced number of GFP-positive cells at the 22-somite stage. Nine control DMSO-treated and 15 SU5402-treated embryos were analyzed in two replicate experiments. gi Heat-shock inducible expression of dnFGFR1 results in a significant reduction of mCherry-positive muscle cells in etv2ci32Gt+/−; UAS:NTR-mCherry; hsp70:dnFGFR1 embryos. Twenty each control and HS hsp70:dnFGFR1 embryos were analyzed in two replicate experiments. Median ± SD values are shown in all graphs, two-tailed Student’s t test was used for statistical analysis.

Fig. 7 Expression of LPM cluster genes partially overlaps with <italic>etv2</italic><sup><italic>ci32Gt</italic></sup><italic>; UAS:GFP</italic> expression.

al Fluorescent in situ hybridization using hybridization chain reaction for prrx1a (af) and twist1a (gl) expression combined with GFP fluorescence in etv2ci32Gt; UAS:GFP heterozygous or homozygous embryos at the 18-somite stage. Maximum intensity projections of selected confocal z-stacks are shown. Note that both prrx1a and twist1a are expressed bilaterally, and their expression partially overlaps with GFP in the most lateral cells (arrows). GFP+ cells fail to coalesce into axial vasculature in etv2ci32Gt−/− embryos. DA precursor vessel for the dorsal aorta, LDA lateral dorsal aortae. m, n Expression of foxd2 in the trunk region in etv2ci32Gt heterozygous and homozygous embryos at the 18-somite stage. Note expression in the LPM region (arrows). Expression in the somites is also apparent. o, p In situ hybridization for GFP expression in etv2ci32Gt+/− and etv2ci32Gt−/−; UAS:GFP embryos at the 17–18-somite stage. GFP expression is observed in endothelial cell (EC, combined EC1 and EC2), endocardial (Endoc), endothelial progenitor cell (EPC), lateral plate mesoderm (LPM), tailbud and red blood cell (RBC) populations. Note the absence of EC and endocardial expression in (p). In all panels, the numbers display the number of embryos showing the expression pattern out of the total number of embryos analyzed in two replicate experiments.

Fig. 8 Single-cell RNA-seq analysis using Fluidigm cell sorting of <italic>Tg(-2.3 etv2:GFP)</italic> embryos at the 16–20-somite stage.

ac GFP expression in live embryos, maximum intensity projection is shown. Arrows label vascular endothelial cells and their progenitors. Ten embryos were imaged in two independent experiments and a representative embryo is shown. a lateral view; b anterior view, c dorsal view. A anterior, P posterior. d Heatmap view of marker gene expression in different cell clusters. A complete list of differentially expressed genes is presented in Supplementary Data 5. e, f 2-D and 3-D principal component analysis plots of different cell clusters. Cluster names are the same as in (d). g Relative marker gene expression in different cell clusters. Vertical bars depict log-normalized gene expression. h An Arteriovenous (A-V) index of different endothelial cells. Note that many cells are positive for both arterial and venous marker expression. il ISH expression analysis of key marker genes for EPC (etv2), venous (flt4), arterial (cldn5b), and EC-2 (ldb2a) populations in the trunk region at the 20-somite stage. Black arrows label the DA and white arrowheads label venous progenitors which are starting to coalesce into the PCV. Note that flt4 is enriched in the PCV while cldn5b and ldb2a label the DA. mr Two color ISH analysis for the expression of venous dab2 and arterial cldn5b at the 20-somite and 24 hpf stages. Arrows label the DA while arrowheads mark the PCV or its progenitors. Note that dab2 and cldn5b are co-expressed in the DA progenitors at the 20-somite stage but not at 24 hpf. In all panels, the numbers in the lower right corner display the number of embryos showing the expression pattern out of the total number of embryos analyzed in two replicate experiments.

Fig. 9 A proposed model for the differentiation of vascular endothelial cells from the multipotent mesodermal progenitors in the LPM.

Wnt and FGF signaling promotes myocyte differentiation of multipotent progenitors in the lateral plate mesoderm (LPM). BMP signaling through its downstream effectors id1 and id3 promotes vascular endothelial differentiation. Additional LPM markers that include prrx1a, foxd1, foxd2 and others may be involved in this process, although their role is purely speculative at this point. Etv2 promotes vascular endothelial differentiation while directly or indirectly repressing myogenesis.

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