During vertebrate embryogenesis, hematopoietic stem cells (HSC) arise in the aorta-gonads-mesonephros (AGM) region. A zebrafish chemical genetic screen identified compounds that regulate blood flow as modulators of HSC formation. silent heart (sih) embryos that lack a heartbeat and blood circulation exhibited severely reduced HSCs. Blood flow modifiers exerted their effects after the onset of heartbeat; however, nitric oxide (NO) donors affected HSC induction even when treatment occurred prior to the initiation of circulation, and rescued HSCs in sih mutants. NO synthase (Nos) inhibitors and morpholino-knockdown of nos1 (nnos/enos) blocked HSC development. Embryonic transplantation assays demonstrated a cell-autonomous requirement for nos1. Nos3 (eNos) was expressed in HSCs in the murine AGM. Intrauterine Nos inhibition or Nos3 deficiency in mice resulted in the absence of hematopoietic clusters and reduced transplantable progenitors and HSCs. This work links blood flow to AGM hematopoiesis, and identifies NO as a conserved downstream regulator of HSC development.

Summary Hematopoietic stem cells (HSCs) are used in transplantation therapy to reconstitute the hematopoietic system. Human cord blood (hCB) transplantation has emerged as an attractive alternative treatment option when traditional HSC sources are unavailable, however, the absolute number of hCB HSCs transplanted is significantly lower than bone marrow or mobilized peripheral blood stem cells (MPBSCs). We previously demonstrated that dimethyl-prostaglandin E2 (dmPGE2) increased HSCs in vertebrate models. Here, we describe preclinical analyses of the therapeutic potential of dmPGE2-treatment using human and non-human primate HSCs. dmPGE2 significantly increased total human hematopoietic colony formation in vitro and enhanced engraftment of unfractionated and CD34+ hCB following xenotransplantation. In non-human primate autologous transplantation, dmPGE2-treated CD34+ MPBSCs showed stable multilineage engraftment over one year post-infusion. Together, our analyses indicated that dmPGE2 mediates conserved responses in HSCs from human and non-human primates, and provided sufficient preclinical information to support proceeding to an FDA-approved phase 1 clinical trial.

• Glucose metabolism enhances hematopoietic stem cell formation and function in the vertebrate embryo • Glucose metabolism modulates hif1a activity via mitochondrial generation of reactive oxygen species to impact HSC-relevant gene expressionMany pathways regulating blood formation have been elucidated, yet how each coordinates with embryonic biophysiology to modulate the spatiotemporal production of hematopoietic stem cells (HSCs) is currently unresolved. Here, we report that glucose metabolism impacts the onset and magnitude of HSC induction in vivo. In zebrafish, transient elevations in physiological glucose levels elicited dose-dependent effects on HSC development, including enhanced runx1 expression and hematopoietic cluster formation in the aorta-gonad-mesonephros region; embryonic-to-adult transplantation studies confirmed glucose increased functional HSCs. Glucose uptake was required to mediate the enhancement in HSC development; likewise, metabolic inhibitors diminished nascent HSC production and reversed glucose-mediated effects on HSCs. Increased glucose metabolism preferentially impacted hematopoietic and vascular targets, as determined by gene expression analysis, through mitochondrialderived reactive oxygen species (ROS)-mediated stimulation of hypoxia-inducible factor 1a (hif1a). Epistasis assays demonstrated that hif1a regulates HSC formation in vivo and mediates the dose-dependent effects of glucose metabolism on the timing and magnitude of HSC production. We propose that this fundamental metabolic-sensing mechanism enables the embryo to respond to changes in environmental energy input and adjust hematopoietic output to maintain embryonic growth and ensure viability. (Blood. 2013;121(13):2483-2493

Genetic mutations responsible for oblique facial clefts (ObFC), a unique class of facial malformations, are largely unknown. We show that loss-of-function mutations in SPECC1L are pathogenic for this human developmental disorder and that SPECC1L is a critical organizer of vertebrate facial morphogenesis. During murine embryogenesis, Specc1l is expressed in cell populations of the developing facial primordial, which proliferate and fuse to form the face. In zebrafish, knockdown of a SPECC1L homolog produces a faceless phenotype with loss of jaw and facial structures, and knockdown in Drosophila phenocopies mutants in the integrin signaling pathway that exhibit cell-migration and -adhesion defects. Furthermore, in mammalian cells, SPECC1L colocalizes with both tubulin and actin, and its deficiency results in defective actin-cytoskeleton reorganization, as well as abnormal cell adhesion and migration. Collectively, these data demonstrate that SPECC1L functions in actin-cytoskeleton reorganization and is required for proper facial morphogenesis.