HDACIs increase c-myb⁺ HSPC number and runx1 expression in zebrafish embryos. (a) Schematic representation of HSPC development in the AGM and CHT regions of a zebrafish embryo. YS – yolk sac; YE – yolk extension; DA – dorsal aorta; AV – axial vein; AGM – aorta-gonad-mesonephros; CHT – caudal hematopoietic tissue. Small green circles between the DA and AV and in CHT regions represent HSPCs. (b) Image based identification of c-myb⁺ cells in the AGM and CHT region identified HDACIs (valproic acid, resminostat, and entinostat) as enhancers of HSPC cell-count at 36 hpf (Bars = 200 µm). The region-of-interest (AGM and CHT) marked by white line and the c-myb⁺ cells are marked by red circle (indicated by red arrows) and the false positive objects that are excluded from the quantification are marked by yellow circle (indicated by yellow arrows). (c) Quantification of relative number of c-myb⁺ cells in the AGM and CHT region showing increased cell-count after 40 µM VPA, resminostat or entinostat treatment compared to DMSO (n = 5). (d) Validation of identified hits through whole-mount in situ hybridization for runx1 expression. (e) Quantification of runx1 relative stain intensity shows significantly higher runx1 expression in HDACI treated fish compared to DMSO controls. Intensity was calculated using area under curve analyses in ImageJ. Single images were split into 5 regions of interest (ROIs) and intensity was normalized to background signal. SD displays deviation among 5 ROIs in one image (n = 3). Data are shown as mean ± SD, *p < 0.05; **p < 0.01; *** p < 0.001.