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Summary Hematopoietic stem/progenitor cells (HSPCs) are formed during ontogeny from hemogenic endothelium in the ventral wall of the dorsal aorta (VDA). Critically, the cellular mechanism(s) allowing HSPC egress and migration to secondary niches are incompletely understood. Matrix metalloproteinases (MMPs) are inflammation-responsive proteins that regulate extracellular matrix (ECM) remodeling, cellular interactions, and signaling. Here, inhibition of vascular-associated Mmp2 function caused accumulation of fibronectin-rich ECM, retention of runx1/cmyb+ HSPCs in the VDA, and delayed caudal hematopoietic tissue (CHT) colonization; these defects were absent in fibronectin mutants, indicating that Mmp2 facilitates endothelial-to-hematopoietic transition via ECM remodeling. In contrast, Mmp9 was dispensable for HSPC budding, being instead required for proper colonization of secondary niches. Significantly, these migration defects were mimicked by overexpression and blocked by knockdown of C-X-C motif chemokine-12 (cxcl12), suggesting that Mmp9 controls CHT homeostasis through chemokine regulation. Our findings indicate Mmp2 and Mmp9 play distinct but complementary roles in developmental HSPC production and migration.

Summary Hematopoietic stem and progenitor cells (HSPCs) are born from hemogenic endothelium in the dorsal aorta. Specification of this hematopoietic niche is regulated by a signaling axis using Hedgehog (Hh) and Notch, which culminates in expression of Runx1 in the ventral wall of the artery. Here, we demonstrate that the vitamin D precursor cholecalciferol (D3) modulates HSPC production by impairing hemogenic vascular niche formation. Accumulation of D3 through exogenous treatment or inhibition of Cyp2r1, the enzyme required for D3 25-hydroxylation, results in Hh pathway antagonism marked by loss of Gli-reporter activation, defects in vascular niche identity, and reduced HSPCs. Mechanistic studies indicated the effect was specific to D3, and not active 1,25-dihydroxy vitamin D3, acting on the extracellular sterol-binding domain of Smoothened. These findings highlight a direct impact of inefficient vitamin D synthesis on cell fate commitment and maturation in Hh-regulated tissues, which may have implications beyond hemogenic endothelium specification.

Zebrafish are one of the preeminent model systems for the study of blood development (hematopoiesis), hematopoietic stem and progenitor cell (HSPC) biology, and hematopathology. The zebrafish hematopoietic system shares strong similarities in functional populations, genetic regulators, and niche interactions with its mammalian counterparts. These evolutionarily conserved characteristics, together with emerging technologies in live imaging, compound screening, and genetic manipulation, have been employed to successfully identify and interrogate novel regulatory mechanisms and molecular pathways that guide hematopoiesis. Significantly, perturbations in many of the key developmental signals controlling hematopoiesis are associated with hematological disorders and disease, including anemia, bone marrow failure syndromes, and leukemia. Thus, understanding the regulatory pathways controlling HSPC production and function has important clinical implications. In this review, we describe how the blood system forms and is maintained in zebrafish, with particular focus on new insights into vertebrate hematological regulation gained using this model. The interplay of factors controlling development and disease in the hematopoietic system combined with the unique attributes of the zebrafish make this a powerful platform to discover novel targets for the treatment of hematological disease., (© 2017 Elsevier Inc. All rights reserved.)

The stepwise progression of common endoderm progenitors into differentiated liver and pancreas organs is regulated by a dynamic array of signals that are not well understood. The nuclear receptor subfamily 5, group A, member 2 gene nr5a2, also known as Liver receptor homolog-1 (Lrh-1) is expressed in several tissues including the developing liver and pancreas. Here, we interrogate the role of Nr5a2 at multiple developmental stages using genetic and chemical approaches and uncover novel pleiotropic requirements during zebrafish liver and pancreas development. Zygotic loss of nr5a2 in a targeted genetic null mutant disrupted the development of the exocrine pancreas and liver, while leaving the endocrine pancreas intact. Loss of nr5a2 abrogated exocrine pancreas markers such as trypsin, while pancreas progenitors marked by ptf1a or pdx1 remained unaffected, suggesting a role for Nr5a2 in regulating pancreatic acinar cell differentiation. In the developing liver, Nr5a2 regulates hepatic progenitor outgrowth and differentiation, as nr5a2 mutants exhibited reduced hepatoblast markers hnf4α and prox1 as well as differentiated hepatocyte marker fabp10a. Through the first in vivo use of Nr5a2 chemical antagonist Cpd3, the iterative requirement for Nr5a2 for exocrine pancreas and liver differentiation was temporally elucidated: chemical inhibition of Nr5a2 function during hepatopancreas progenitor specification was sufficient to disrupt exocrine pancreas formation and enhance the size of the embryonic liver, suggesting that Nr5a2 regulates hepatic vs. pancreatic progenitor fate choice. Chemical inhibition of Nr5a2 at a later time during pancreas and liver differentiation was sufficient to block the formation of mature acinar cells and hepatocytes. These findings define critical iterative and pleiotropic roles for Nr5a2 at distinct stages of pancreas and liver organogenesis, and provide novel perspectives for interpreting the role of Nr5a2 in disease., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Umbilical cord blood (UCB) is a valuable source of hematopoietic stem cells (HSCs) for use in allogeneic transplantation. Key advantages of UCB are rapid availability and less stringent requirements for HLA matching. However, UCB contains an inherently limited HSC count, which is associated with delayed time to engraftment, high graft failure rates, and early mortality. 16,16-Dimethyl prostaglandin E2 (dmPGE2) was previously identified to be a critical regulator of HSC homeostasis, and we hypothesized that brief ex vivo modulation with dmPGE2 could improve patient outcomes by increasing the "effective dose" of HSCs. Molecular profiling approaches were used to determine the optimal ex vivo modulation conditions (temperature, time, concentration, and media) for use in the clinical setting. A phase 1 trial was performed to evaluate the safety and therapeutic potential of ex vivo modulation of a single UCB unit using dmPGE2 before reduced-intensity, double UCB transplantation. Results from this study demonstrated clear safety with durable, multilineage engraftment of dmPGE2-treated UCB units. We observed encouraging trends in efficacy, with accelerated neutrophil recovery (17.5 vs 21 days, P = .045), coupled with preferential, long-term engraftment of the dmPGE2-treated UCB unit in 10 of 12 treated participants.

Many 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 mitochondrial-derived reactive oxygen species (ROS)-mediated stimulation of hypoxia-inducible factor 1α (hif1α). Epistasis assays demonstrated that hif1α 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.

Growth Factor Independence (Gfi) transcription factors play essential roles in hematopoiesis, differentially activating and repressing transcriptional programs required for hematopoietic stem/progenitor cell (HSPC) development and lineage specification. In mammals, Gfi1a regulates hematopoietic stem cells (HSC), myeloid and lymphoid populations, while its paralog, Gfi1b, regulates HSC, megakaryocyte and erythroid development. In zebrafish, gfi1aa is essential for primitive hematopoiesis; however, little is known about the role of gfi1aa in definitive hematopoiesis or about additional gfi factors in zebrafish. Here, we report the isolation and characterization of an additional hematopoietic gfi factor, gfi1b. We show that gfi1aa and gfi1b are expressed in the primitive and definitive sites of hematopoiesis in zebrafish. Our functional analyses demonstrate that gfi1aa and gfi1b have distinct roles in regulating primitive and definitive hematopoietic progenitors, respectively. Loss of gfi1aa silences markers of early primitive progenitors, scl and gata1. Conversely, loss of gfi1b silences runx-1, c-myb, ikaros and cd41, indicating that gfi1b is required for definitive hematopoiesis. We determine the epistatic relationships between the gfi factors and key hematopoietic transcription factors, demonstrating that gfi1aa and gfi1b join lmo2, scl, runx-1 and c-myb as critical regulators of teleost HSPC. Our studies establish a comparative paradigm for the regulation of hematopoietic lineages by gfi transcription factors., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Developmental signals determine organ morphology and position during embryogenesis. To discover novel modifiers of liver development, we performed a chemical genetic screen in zebrafish and identified retinoic acid as a positive regulator of hepatogenesis. Knockdown of the four RA receptors revealed that all receptors affect liver formation, however specific receptors exert differential effects. Rargb knockdown results in bilateral livers but does not impact organ size, revealing a unique role for Rargb in conferring left-right positional information. Bilateral populations of hepatoblasts are detectable in rargb morphants, indicating Rargb acts during hepatic specification to position the liver, and primitive endoderm is competent to form liver on both sides. Hearts remain at the midline and gut looping is perturbed in rargb morphants, suggesting Rargb affects lateral plate mesoderm migration. Overexpression of Bmp during somitogenesis similarly results in bilateral livers and midline hearts, and inhibition of Bmp signaling rescues the rargb morphant phenotype, indicating Rargb functions upstream of Bmp to regulate organ sidedness. Loss of rargb causes biliary and organ laterality defects as well as asplenia, paralleling symptoms of the human condition right atrial isomerism. Our findings uncover a novel role for RA in regulating organ laterality and provide an animal model of one form of human heterotaxia., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Developmental signaling pathways hold the keys to unlocking the promise of adult tissue regeneration, and to inhibiting carcinogenesis. Patients with mutations in the Adenomatous Polyposis Coli (APC) gene are at increased risk of developing hepatoblastoma, an embryonal form of liver cancer, suggesting that Wnt affects hepatic progenitor cells. To elucidate the role of APC loss and enhanced Wnt activity in liver development, we examined APC mutant and wnt inducible transgenic zebrafish. APC(+/-) embryos developed enlarged livers through biased induction of hepatic gene programs and increased proliferation. Conversely, APC(-/-) embryos formed no livers. Blastula transplantations determined that the effects of APC loss were cell autonomous. Induction of wnt modulators confirmed biphasic consequences of wnt activation: endodermal pattern formation and gene expression required suppression of wnt signaling in early somitogenesis; later, increased wnt activity altered endodermal fate by enhancing liver growth at the expense of pancreas formation; these effects persisted into the larval stage. In adult APC(+/-) zebrafish, increased wnt activity significantly accelerated liver regeneration after partial hepatectomy. Similarly, liver regeneration was significantly enhanced in APC(Min/+) mice, indicating the conserved effect of Wnt pathway activation in liver regeneration across vertebrate species. These studies reveal an important and time-dependent role for wnt signaling during liver development and regeneration.