Hypoxic Stress-Induced Serotonergic Stimulation Controls Hematopoietic Stem Cell Production Via the Hypothalamic-Pituitary-Interrenal Axis and Glucocorticoid Signaling

There is increasing evidence that neuronal signals contribute to adult hematopoietic stem cell (HSC) production and maintenance; however, the mechanisms by which the central nervous system regulates HSCs and/or the HSC niche and its functional necessity during vertebrate HSC development remains unclear. Serotonin (5HT), a neurotransmitter in the brain and a hormone peripherally, was identified in a screen for HSC modulators in zebrafish. Exposure to 5HT or fluoxetine (5HT reuptake inhibitor) increased hematopoietic stem and progenitor cell (HSPC) formation in the zebrafish aorta-gonad-mesonephros (AGM) region as shown by runx1/cmyb in situ hybridization, and confirmed by FACS and qRT-PCR analysis (p<0.05). The stimulatory effect of 5HT was seen both during HSPC niche formation, as well as when AGM HSPCs emerge and expand. Peripheral and neuronal 5HT are synthesized by tryptophan hydroxylase 1 (TPH1) and TPH2, respectively. Chemical inhibition of these rate-limiting enzymes decreased HSC numbers during HSPC emergence (36 hpf). Using morpholino-mediated knockdown, only the effects of selective tph2 reduction on HSCs, and not that of tph1, were found to persist by 48 hpf (p<0.05), which could be rescued by exogenous 5HT; this finding was confirmed using an inducible tph2 -ablation model. Although the sympathetic nervous system (SNS) can contribute to HSC emergence, the effect of 5HT was independent of SNS function as shown using 6-hydroxydopamine (6-OHDA) to induce sympathetic nerve lesions. 5HT can activate the hypothalamic-pituitary-adrenal (HPA) axis, leading to cortisol production and activation of glucocorticoid receptor (GR) to mediate peripheral responses. Indeed, mRNA levels of corticotropin-releasing hormone receptor, pro-opiomelanocortin (pomc) and nr3c1 (GR), as well as cortisol levels were increased in 5HT-treated embryos (p<0.05). In contrast, cortisol production was decreased in tph2 morphants (p<0.05). Consistent with a role for neuronal 5HT in regulating HSCs through the Hypothalamic-Pituitary-Interrenal (HPI) axis (teleost HPA axis counterpart) and peripheral cortisol production, exposure to GR agonists hydrocortisone and dexamethasone increased embryonic HSPC production, while MO-knockdown of GR or analysis of GR mutants revealed drastic reductions in HSPCs and rag2+ lymphoid progenitors. Further, epistasis analysis revealed the impact of 5HT or GR agonist on HSPCs was abrogated upon MO knockdown of GR, whereas GR agonist treatment rescued HSPC numbers in corticotropin-releasing hormone and pomc morphants (p<0.05). The HPA/I axis is well recognized as a "stress response" pathway; we, and others, previously showed that local hypoxia and hypoxic stress-induced Hypoxia Inducible Factor 1a (HIF1a) activity play a pivotal role during embryonic HSPC development. We therefore examined whether hypoxic stimulation can initiate serotonergic and HPI Axis-mediated regulation of HSPC production. Consistent with previous reports, HIF1a activation by cobalt (II) chloride (CoCl₂) increased runx1 + HSPCs in the AGM; CoCl₂ exposure also elevated tph2 (but not tph1) and HPI axis gene expression and total cortisol production (p<0.05). Furthermore, the effect of CoCl₂ stimulation on AGM HSPCs is blocked in tph2 morphants (p<0.05), suggesting that HIF1a activates TPH2-mediated 5HT production to impact HSPC number. Collectively, our data highlight a novel regulatory mechanism controlling embryonic HSPC induction and expansion that exploits the hypoxic stress response to induce serotonergic stimulation of the HPI axis and glucocorticoid signaling.