Figure 6. Hypothalamic-Pituitary-Interrenal Axis and Glucocorticoid Receptor Activation Are Required for Serotonin Stimulation of HSPCs.

(A) Effect of serotonin (7μM) and hydrocortisone (25μM) treatment on runx1 WISH in control or nr3c1 MO-injected embryos at 30hpf.

(B) Qualitative phenotype distribution graph of (A).

(C) Flk1:dsRed⁺cMyb:GFP⁺ HSPCs were significantly increased in serotonin- and hydrocortisone-treated WT embryos, but not in nr3c1 morphants, at 48hpf by FACS (n≥15 replicates; mean±SD; two-way ANOVA, Holm-Sidak post hoc: *p<0.05, ****p<0.0001).

(D) GR mutant (GR^s357) embryos had dramatically decreased runx1 expression in the AGM by WISH at 30hpf, which could not be rescued by serotonin (7μM).

(E) Qualitative phenotype distribution graph of (D).

(F) Altered WISH for cmyb⁺ HSPCs in the CHT at 72hpf and rag2⁺ lymphoid cells at 5dpf in the thymus in GR mutant embryos compared to controls.

(G) Qualitative phenotype distribution graph of (F).

(H) Representative images of decreased CD41⁺ cells in runx1:nr3c1-injected Tg(−6.0itga2b:egfp) embryos at 48hpf.

(I) Cell count of (H); n≥8; mean±SD; two-tailed t-test: **p<0.01.

(J) Injection of runx1:nr3c1 in GR^s357 embryos rescued runx1⁺ HSPCs at 30hpf by WISH.

(K) MK212 (7.5μM) could not rescue HSPC defects seen in crh and pomc MO-injected embryos at 30hpf by runx1 WISH.

(L) Qualitative phenotype distribution graph of (J).

(M) FACS analysis of (L) with Flk1:dsRed⁺cMyb:GFP⁺ HSPCs at 48hpf by FACS (n≥3 replicates; mean±SD; two-way ANOVA, Holm-Sidak post hoc: *p<0.05).

Scale bars=100μM.

See also Figure S7.