(A) Sequence alignment of human SHP2 and zebrafish Shp2a polypeptides performed by ClustalW. The residue D61 is highlighted in red, while the identical amino acids are highlighted in black. The amino acid number is marked to the right of each row. (B) Schematic representation of the procedure used to establish the Shp2^D61G mutant line, as described in the Materials and methods section. (C) Representative images of the WISH staining for myl7, ahmc, and vhmc expression in the hearts of 3 days post fertilization (dpf) old Shp2^wt, Shp2^D61G/wt, and Shp2^D61G/D61G zebrafish embryos. Numbers in the pictures indicate the number of embryos with the phenotype represented in the image. Ejection fraction (D) and cardiac output (E) determined from the high-speed videos of the heart of 5 dpf old Shp2^wt, Shp2^D61G/wt, and Shp2^D61G/D61G zebrafish embryos. Measurements originate from at least three distinct experiments. Numbers on the bars depict the number of embryos. Error bars represent standard error of the mean (SEM).*p < 0.05; **p < 0.01, ***p < 0.001, NS, non-significant, ANOVA complemented by Tukey’s HSD.

(A) Representative images of the whole-mount in situ hybridization (WISH) staining for gata1, pu.1, and l-plastin expression in the tail region of 48 hr post fertilization (hpf) old Shp2^wt, Shp2^D61G/wt, and Shp2^D61G/D61G zebrafish embryos. Scale bar, 250 µm. Numbers in the pictures indicate the number of embryos with the phenotype represented in the image. (B) Cell proliferation was assessed in the caudal hematopoietic tissue (CHT) region of 5 days post fertilization (dpf) old Shp2^wt, Shp2^D61G/wt, and Shp2^D61G/D61G embryos in the Tg(cd41:GFP) background by immunohistochemistry using antibodies specific for phosphohistone H3 (pHis3) and GFP. Cells positive for both GFP and pHis3 are indicated with white arrows in the merge panel. Scale bar, 10 μm. (C) Representative images of the Acridine orange staining of the CHT region of the 5 dpf old Shp2^wt, Shp2^D61G/wt, and Shp2^D61G/D61G embryos. Scale bar, 50 μm. (D) WISH of 5 dpf Shp2^wt, Shp2^D61G/wt, and Shp2^D61G/D61G embryos using ikaros-specific probe. Thymus is indicated (arrow). (E) Size of ikaros-positive thymus, NS, non-significant, ANOVA complemented by Tukey’s HSD. (F) Number of cd41:GFP^high cells in Shp2^wt, Shp2^D61G/wt, and Shp2^D61G/D61G zebrafish embryos at 5 dpf were counted and percentage of embryos with either 0–50, 50–75, or 75–100 cd41:GFP^high cells was plotted. (G) WISH of 5 dpf Shp2^wt, Shp2^D61G/wt, and Shp2^D61G/D61G embryos using β-globin-specific probe. Scale bar, 150 μm. (H) Quantification of β-globin expression in Shp2^wt, Shp2^D61G/wt, and Shp2^D61G/D61G embryos scored as low, mid, and high. (E,F,H) Measurements originate from at least three distinct experiments. Numbers on the bars depict the number of embryos.

(A) Heat map showing scaled expression (log TPM [transcripts per million] values) of representative genes (y-axis) in all cells (x-axis). Cells belonging to distinct clusters are squared with a dashed line and cluster identity is indicated on the top of the heat map. Cluster 1 is characterized by expression of genes characteristic for thrombocyte and erythroid differentiation, such as gata1a and klf1. Cells from cluster 2 express typical hematopoietic stem cell markers, such as c-myb and her6 (HES1 analogue in human). Cells from cluster 3 express early myeloid progenitor markers, such as runx3 and cxcr4b. Cells from cluster 4 express genes typical for both monocyte and macrophages, such as marco and ctsba. (B) t-Distributed stochastic neighbor embedding (t-SNE) maps showing the sum of total read-counts of selected neutrophil-specific genes.

(A) Identities of the four clusters of HSPCs of Shp2^wt, Shp2^D61G/wt and Shp2^D61G/D61G genotypes from the trajectory in Figure 4, ordered in pseudotime. (B) Heat map showing scaled expression (log TPM [transcripts per million] values) of representative genes (y-axis) in cells of Shp2^wt, Shp2^D61G/wt, and Shp2^D61G/D61G genotypes from the selected trajectory route, ordered along the pseudotime (x-axis). The cell type and pseudotime are annotated above the heat map.