ism1 is highly expressed in hematopoietic-supportive stromal cell lines.

(A) Each stromal cell line is derived from sites of hematopoietic emergence, expansion, and maintenance in adult and embryonic zebrafish; adult zebrafish kidney stroma (ZKS, derived from the adult kidney; pink[50]), zebrafish embryonic stromal trunk (ZEST, derived from the embryonic trunk; blue[48]), and caudal hematopoietic embryonic stromal tissue (CHEST, derived from the CHT; green[49]). (B) Each stromal cell line’s transcriptome was analyzed with RNA-seq and compared to a non-hematopoietic supportive stromal cell line (ZF4); transcripts were plotted as a Venn diagram demonstrating 447 conserved transcripts. (C) ism1 transcript levels for each cell line presented as reads per kilobase of transcript per million reads mapped (RPKM). See Table 1 for genes identified in this screen.

ism1 knockdown decreases erythrocytes.

gata1:DsRed zebrafish embryos were injected at the single-cell-stage with 7 ng ism1 MO (MO), or 7 ng ism1 MO and 17.88 ng of ism1 mRNA (Rescue); uninjected embryos served as controls. (A) 24 hpf zebrafish visualized at 5x that have normal (left), pooled blood (middle), and slowed circulation (right) phenotypes. (B) Quantitation of zebrafish shown in (A); pooled blood (yellow), slowed circulation (blue), and normal (red) phenotypes. (C) Flow cytometry results quantitating gata1:DsRed⁺ erythrocytes in uninjected (control; red), ism1 MO injected (MO; peach), and ism1 MO with ism1 mRNA (Rescue; pink) 72 hpf zebrafish. (D) qRT-PCR was performed comparing transcript levels of hbaa in ism1 MO injected (MO) and uninjected controls (control). Bars represent the mean, and error bars represent standard deviation. Data was normalized as fold change over control and analyzed using a two-tailed unpaired Student’s T test. * denotes p<0.05, ** denotes p<0.0001, N.S indicates no significance.

ism1 knockdown decreases neutrophils and macrophages in 48 hpf zebrafish.

mpx:GFP single-cell-stage embryos were injected with 7 ng ism1 MO (MO; squares), or 7 ng ism1 MO and 17.88 ng of ism1 mRNA (Rescue; triangles); uninjected embryos served as controls (circles). (A) Developing 48 hpf zebrafish were visualized, and individual mpx:GFP⁺ cells were enumerated; representative fish shown for reference. (B) Each data point represents total amount of mpx:GFP⁺ cells present in one zebrafish. (C) qRT-PCR was performed comparing transcript levels of csf3r (left) and csf1r (right) in ism1 MO injected (MO) and uninjected controls (control) at 48 hpf. Bars represent the mean, and error bars represent standard deviation. A two-tailed unpaired Student’s T test was performed to determine statistical significance. * denotes p<0.05, ** denotes p<0.0001, N.S indicates no significance.

Hematopoietic stem and progenitor cells (HSPCs) are reduced in ism1 knockdown embryos.

Single-cell-stage embryos were injected with 7 ng of ism1 MO (MO), or 7 ng of ism1 MO and 17.88 ng of ism1 mRNA (Rescue); uninjected embryos served as a control. (A) Ten 48 hpf embryos from each treatment were digested, plated in methylcellulose with exogenous hematopoietic-supportive growth factors, and incubated for 7 days. (B) Colony forming units (CFUs) generated by ism1 MO (light blue) and ism1 MO with ism1 mRNA (Rescue; darker blue) injected embryos presented as fold change over control embryos (dark blue). (C) qRT-PCR was performed comparing transcript levels of cmyb and runx1 in ism1 MO injected (MO) and uninjected controls (control) at 48 hpf. Bars represent the mean, and error bars represent standard deviation. A two-tailed unpaired Student’s T test was performed to determine statistical significance. * denotes p<0.05, ** denotes p<0.0001, N.S indicates no significance.

ism1 knockdown leads to temporally shortened intersegmental vessels.

flk1:GFP single-cell-stage embryos were injected with 7 ng of ism1 MO (bottom); uninjected embryos served as controls (top). 24 hpf (left column) and 48 hpf (right column) zebrafish were visualized at 40x for flk1:GFP fluorescence within the trunk area denoted by black box in brightfield image at top center. Arrows indicate shortened intersegmental vessels in ism1 morphants. Numbers in corner of images denote the number of embryos displaying the imaged phenotype.