Synergistic prostaglandin E synthesis by myeloid and endothelial cells promotes fetal hematopoietic stem cell expansion in vertebrates.

During development, hematopoietic stem cells (HSCs) are produced from the hemogenic endothelium and will expand in a transient hematopoietic niche. Prostaglandin E2 (PGE2) is essential during vertebrate development and HSC specification, but its precise source in the embryo remains elusive. Here, we show that in the zebrafish embryo, PGE2 synthesis genes are expressed by distinct stromal cell populations, myeloid (neutrophils, macrophages), and endothelial cells of the caudal hematopoietic tissue. Ablation of myeloid cells, which produce the PGE2 precursor prostaglandin H2 (PGH2), results in loss of HSCs in the caudal hematopoietic tissue, which could be rescued by exogeneous PGE2 or PGH2 supplementation. Endothelial cells contribute by expressing the PGH2 import transporter slco2b1 and ptges3, the enzyme converting PGH2 into PGE2. Of note, differential niche cell expression of PGE2 biosynthesis enzymes is also observed in the mouse fetal liver. Taken altogether, our data suggest that the triad composed of neutrophils, macrophages, and endothelial cells sequentially and synergistically contributes to blood stem cell expansion during vertebrate development.

Notch signaling enhances bone regeneration in the zebrafish mandible.

Loss or damage to the mandible caused by trauma, treatment of oral malignancies, and other diseases is treated using bone-grafting techniques that suffer from numerous shortcomings and contraindications. Zebrafish naturally heal large injuries to mandibular bone, offering an opportunity to understand how to boost intrinsic healing potential. Using a novel her6:mCherry Notch reporter, we show that canonical Notch signaling is induced during the initial stages of cartilage callus formation in both mesenchymal cells and chondrocytes following surgical mandibulectomy. We also show that modulation of Notch signaling during the initial post-operative period results in lasting changes to regenerate bone quantity one month later. Pharmacological inhibition of Notch signaling reduces the size of the cartilage callus and delays its conversion into bone, resulting in non-union. Conversely, conditional transgenic activation of Notch signaling accelerates conversion of the cartilage callus into bone, improving bone healing. Given the conserved functions of this pathway in bone repair across vertebrates, we propose that targeted activation of Notch signaling during the early phases of bone healing in mammals may both augment the size of the initial callus and boost its ossification into reparative bone.

bif1, a new BMP signaling inhibitor, regulates embryonic hematopoiesis in the zebrafish.

Hematopoiesis maintains the entire blood system, and dysregulation of this process can lead to malignancies (leukemia), immunodeficiencies or red blood cell diseases (anemia, polycythemia vera). We took advantage of the zebrafish model that shares most of the genetic program involved in hematopoiesis with mammals to characterize a new gene of unknown function, si:ch73-299h12.2, which is expressed in the erythroid lineage during primitive, definitive and adult hematopoiesis. This gene, required during primitive and definitive erythropoiesis, encodes a C2H2 zinc-finger protein that inhibits BMP signaling. We therefore named this gene blood-inducing factor 1 and BMP inhibitory factor 1 (bif1). We identified a bif1 ortholog in Sinocyclocheilus rhinocerous, another fish, and in the mouse genome. Both genes also inhibit BMP signaling when overexpressed in zebrafish. In conclusion, we have deorphanized a new zebrafish gene of unknown function: bif1 codes for a zinc-finger protein that inhibits BMP signaling and also regulates primitive erythropoiesis and definitive hematopoiesis.

Yolk sac hematopoiesis: does it contribute to the adult hematopoietic system?

In most vertebrates, the yolk sac (YS) represents the very first tissue where blood cells are detected. Therefore, it was thought for a long time that it generated all the blood cells present in the embryo. This model was challenged using different animal models, and we now know that YS hematopoietic precursors are mostly transient although their contribution to the adult system cannot be excluded. In this review, we aim at properly define the different waves of blood progenitors that are produced by the YS and address the fate of each of them. Indeed, in the last decade, many evidences have emphasized the role of the YS in the emergence of several myeloid tissue-resident adult subsets. We will focus on the development of microglia, the resident macrophages in the central nervous system, and try to untangle the recent controversy about their origin.

tfec controls the hematopoietic stem cell vascular niche during zebrafish embryogenesis.

In mammals, embryonic hematopoiesis occurs in successive waves, culminating with the emergence of hematopoietic stem cells (HSCs) in the aorta. HSCs first migrate to the fetal liver (FL), where they expand, before they seed the bone marrow niche, where they will sustain hematopoiesis throughout adulthood. In zebrafish, HSCs emerge from the dorsal aorta and colonize the caudal hematopoietic tissue (CHT). Recent studies showed that they interact with endothelial cells (ECs), where they expand, before they reach their ultimate niche, the kidney marrow. We identified tfec, a transcription factor from the mitf family, which is highly enriched in caudal endothelial cells (cECs) at the time of HSC colonization in the CHT. Gain-of-function assays indicate that tfec is capable of expanding HSC-derived hematopoiesis in a non-cell-autonomous fashion. Furthermore, tfec mutants (generated by CRISPR/Cas9) showed reduced hematopoiesis in the CHT, leading to anemia. Tfec mediates these changes by increasing the expression of several cytokines in cECs from the CHT niche. Among these, we found kitlgb, which could rescue the loss of HSCs observed in tfec mutants. We conclude that tfec plays an important role in the niche to expand hematopoietic progenitors through the modulation of several cytokines. The full comprehension of the mechanisms induced by tfec will represent an important milestone toward the expansion of HSCs for regenerative purposes.

Ndrg1b and fam49ab modulate the PTEN pathway to control T-cell lymphopoiesis in the zebrafish.

During hematopoiesis, the balance between proliferation, differentiation, and apoptosis is tightly regulated in order to maintain homeostasis. Failure in these processes can ultimately lead to uncontrolled proliferation and leukemia. Phosphatase and tensin homolog (PTEN) is one of the molecular pathways involved in this balance. By opposing PI3-kinases, PTEN inhibits proliferation and promotes differentiation and is thus considered a tumor suppressor. Indeed, PTEN is frequently mutated in many cancers, including leukemias. Loss of PTEN often leads to lymphoid cancers. However, little is known about the molecular events that regulate PTEN signaling during lymphopoiesis. In this study, we used zebrafish to address this. We report that N-myc downstream-regulated gene 1b (ndrg1b) rescues lymphoid differentiation after PTEN inhibition. We also show that a previously uncharacterized gene, fam49ab, inhibits T-cell differentiation, a phenotype that can be rescued by ndrg1b We propose that ndrg1b and fam49ab are 2 new modulators of PTEN signaling that control lymphoid differentiation in the zebrafish thymus.

An Fgf-Shh signaling hierarchy regulates early specification of the zebrafish skull.

The neurocranium generates most of the craniofacial skeleton and consists of prechordal and postchordal regions. Although development of the prechordal is well studied, little is known of the postchordal region. Here we characterize a signaling hierarchy necessary for postchordal neurocranial development involving Fibroblast growth factor (Fgf) signaling for early specification of mesodermally-derived progenitor cells. The expression of hyaluron synthetase 2 (has2) in the cephalic mesoderm requires Fgf signaling and Has2 function, in turn, is required for postchordal neurocranial development. While Hedgehog (Hh)-deficient embryos also lack a postchordal neurocranium, this appears primarily due to a later defect in chondrocyte differentiation. Inhibitor studies demonstrate that postchordal neurocranial development requires early Fgf and later Hh signaling. Collectively, our results provide a mechanistic understanding of early postchordal neurocranial development and demonstrate a hierarchy of signaling between Fgf and Hh in the development of this structure.

Haematopoietic stem cells derive directly from aortic endothelium during development.

A major goal of regenerative medicine is to instruct formation of multipotent, tissue-specific stem cells from induced pluripotent stem cells (iPSCs) for cell replacement therapies. Generation of haematopoietic stem cells (HSCs) from iPSCs or embryonic stem cells (ESCs) is not currently possible, however, necessitating a better understanding of how HSCs normally arise during embryonic development. We previously showed that haematopoiesis occurs through four distinct waves during zebrafish development, with HSCs arising in the final wave in close association with the dorsal aorta. Recent reports have suggested that murine HSCs derive from haemogenic endothelial cells (ECs) lining the aortic floor. Additional in vitro studies have similarly indicated that the haematopoietic progeny of ESCs arise through intermediates with endothelial potential. Here we have used the unique strengths of the zebrafish embryo to image directly the generation of HSCs from the ventral wall of the dorsal aorta. Using combinations of fluorescent reporter transgenes, confocal time-lapse microscopy and flow cytometry, we have identified and isolated the stepwise intermediates as aortic haemogenic endothelium transitions to nascent HSCs. Finally, using a permanent lineage tracing strategy, we demonstrate that the HSCs generated from haemogenic endothelium are the lineal founders of the adult haematopoietic system.

An evolutionarily conserved program of B-cell development and activation in zebrafish.

Teleost fish are among the most ancient vertebrates possessing an adaptive immune system with B and T lymphocytes that produce memory responses to pathogens. Most bony fish, however, have only 2 types of B lymphocytes, in contrast to the 4 types available to mammals. To better understand the evolution of adaptive immunity, we generated transgenic zebrafish in which the major immunoglobulin M (IgM(+)) B-cell subset expresses green fluorescence protein (GFP) (IgM1:eGFP). We discovered that the earliest IgM(+) B cells appear between the dorsal aorta and posterior cardinal vein and also in the kidney around 20 days postfertilization. We also examined B-cell ontogeny in adult IgM1:eGFP;rag2:DsRed animals, where we defined pro-B, pre-B, and immature/mature B cells in the adult kidney. Sites of B-cell development that shift between the embryo and adult have previously been described in birds and mammals. Our results suggest that this developmental shift occurs in all jawed vertebrates. Finally, we used IgM1:eGFP and cd45DsRed;blimp1:eGFP zebrafish to characterize plasma B cells and investigate B-cell function. The IgM1:eGFP reporter fish are the first nonmammalian B-cell reporter animals to be described. They will be important for further investigation of immune cell evolution and development and host-pathogen interactions in zebrafish.

Characterization of the mononuclear phagocyte system in zebrafish.

The evolutionarily conserved immune system of the zebrafish (Danio rerio), in combination with its genetic tractability, position it as an excellent model system in which to elucidate the origin and function of vertebrate immune cells. We recently reported the existence of antigen-presenting mononuclear phagocytes in zebrafish, namely macrophages and dendritic cells (DCs), but have been impaired in further characterizing the biology of these cells by the lack of a specific transgenic reporter line. Using regulatory elements of a class II major histocompatibility gene, we generated a zebrafish reporter line expressing green fluorescent protein (GFP) in all APCs, macrophages, DCs, and B lymphocytes. Examination of mhc2dab:GFP; cd45:DsRed double-transgenic animals demonstrated that kidney mhc2dab:GFP(hi); cd45:DsRed(hi) cells were exclusively mature monocytes/macrophages and DCs, as revealed by morphologic and molecular analyses. Mononuclear phagocytes were found in all hematolymphoid organs, but were most abundant in the intestine and spleen, where they up-regulate the expression of inflammatory cytokines upon bacterial challenge. Finally, mhc2dab:GFP and cd45:DsRed transgenes mark mutually exclusive cell subsets in the lymphoid fraction, enabling the delineation of the major hematopoietic lineages in the adult zebrafish. These findings suggest that mhc2dab:GFP and cd45:DsRed transgenic lines will be instrumental in elucidating the immune response in the zebrafish.

Minichromosome maintenance protein 10 (mcm10) regulates hematopoietic stem cell emergence in the zebrafish embryo.

Hematopoietic stem cells (HSCs) guarantee the continuous supply of all blood lineages during life. In response to stress, HSCs are capable of extensive proliferative expansion, whereas in steady state, HSCs largely remain in a quiescent state to prevent their exhaustion. DNA replication is a very complex process, where many factors need to exert their functions in a perfectly concerted manner. Mini-chromosome-maintenance protein 10 (Mcm10) is an important replication factor, required for proper assembly of the eukaryotic replication fork. In this report, we use zebrafish to study the role of mcm10 during embryonic development, and we show that mcm10 specifically regulates HSC emergence from the hemogenic endothelium. We demonstrate that mcm10-deficient embryos present an accumulation of DNA damages in nascent HSCs, inducing their apoptosis. This phenotype can be rescued by knocking down p53. Taken all together, our results show that mcm10 plays an important role in the emergence of definitive hematopoiesis.

Notch signaling distinguishes 2 waves of definitive hematopoiesis in the zebrafish embryo.

Recent studies have revealed that definitive hematopoiesis in vertebrates initiates through the formation of a non-self-renewing progenitor with limited multilineage differentiation potential termed the erythromyeloid progenitor (EMP). EMPs are specified before hematopoietic stem cells (HSCs), which self-renew and are capable of forming all mature adult blood lineages including lymphoid cells. Despite their differences, EMPs and HSCs share many phenotypic traits, making precise study of their respective functions difficult. Here, we examine whether embryonic specification of EMPs requires Notch signaling as has been shown for HSCs. In mindbomb mutants, which lack functional Notch ligands, we show that EMPs are specified normally: we detect no significant differences in cell number, gene expression, or differentiation capacity between EMPs purified from wild-type (WT) or mindbomb mutant embryos. Similarly N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT), a chemical inhibitor of Notch receptor activation, has no effect on EMP specification. These studies establish that HSCs are the only hematopoietic precursor that requires Notch signaling and help to clarify the signaling events underlying the specification of the 2 distinct waves of definitive hematopoiesis.

Mutations in the cilia gene ARL13B lead to the classical form of Joubert syndrome.

Joubert syndrome (JS) and related disorders are a group of autosomal-recessive conditions sharing the "molar tooth sign" on axial brain MRI, together with cerebellar vermis hypoplasia, ataxia, and psychomotor delay. JS is suggested to be a disorder of cilia function and is part of a spectrum of disorders involving retinal, renal, digital, oral, hepatic, and cerebral organs. We identified mutations in ARL13B in two families with the classical form of JS. ARL13B belongs to the Ras GTPase family, and in other species is required for ciliogenesis, body axis formation, and renal function. The encoded Arl13b protein was expressed in developing murine cerebellum and localized to the cilia in primary neurons. Overexpression of human wild-type but not patient mutant ARL13B rescued the Arl13b scorpion zebrafish mutant. Thus, ARL13B has an evolutionarily conserved role mediating cilia function in multiple organs.

A connexin/ifi30 pathway bridges HSCs with their niche to dampen oxidative stress.

Reactive oxygen species (ROS) represent a by-product of metabolism and their excess is toxic for hematopoietic stem and progenitor cells (HSPCs). During embryogenesis, a small number of HSPCs are produced from the hemogenic endothelium, before they colonize a transient organ where they expand, for example the fetal liver in mammals. In this study, we use zebrafish to understand the molecular mechanisms that are important in the caudal hematopoietic tissue (equivalent to the mammalian fetal liver) to promote HSPC expansion. High levels of ROS are deleterious for HSPCs in this niche, however this is rescued by addition of antioxidants. We show that Cx41.8 is important to lower ROS levels in HSPCs. We also demonstrate a new role for ifi30, known to be involved in the immune response. In the hematopoietic niche, Ifi30 can recycle oxidized glutathione to allow HSPCs to dampen their levels of ROS, a role that could be conserved in human fetal liver.

Hapln1b, a central organizer of the ECM, modulates kit signaling to control developmental hematopoiesis in zebrafish.

During early vertebrate development, hematopoietic stem and progenitor cells (HSPCs) are produced in hemogenic endothelium located in the dorsal aorta, before they migrate to a transient niche where they expand to the fetal liver and the caudal hematopoietic tissue, in mammals and zebrafish, respectively. In zebrafish, previous studies have shown that the extracellular matrix (ECM) around the aorta must be degraded to enable HSPCs to leave the aortic floor and reach blood circulation. However, the role of the ECM components in HSPC specification has never been addressed. In this study, hapln1b, a key component of the ECM, was specifically expressed in hematopoietic sites in the zebrafish embryo. Gain- and loss-of-function experiments all resulted in the absence of HSPCs in the early embryo, showing that hapln1b is necessary, at the correct level, to specify HSPCs in the hemogenic endothelium. Furthermore, the expression of hapln1b was necessary to maintain the integrity of the ECM through its link domain. By combining functional analyses and computer modeling, we showed that kitlgb interacts with the ECM to specify HSPCs. The findings show that the ECM is an integral component of the microenvironment and mediates the cytokine signaling that is necessary for HSPC specification.

Developmental and tissue-specific expression of NITRs.

Novel immune-type receptors (NITRs) are encoded by large multi-gene families and share structural and signaling similarities to mammalian natural killer receptors (NKRs). NITRs have been identified in multiple bony fish species, including zebrafish, and may be restricted to this large taxonomic group. Thirty-nine NITR genes that can be classified into 14 families are encoded on zebrafish chromosomes 7 and 14. Herein, we demonstrate the expression of multiple NITR genes in the zebrafish ovary and during embryogenesis. All 14 families of zebrafish NITRs are expressed in hematopoietic kidney, spleen and intestine as are immunoglobulin and T cell antigen receptors. Furthermore, all 14 families of NITRs are shown to be expressed in the lymphocyte lineage, but not in the myeloid lineage, consistent with the hypothesis that NITRs function as NKRs. Sequence analyses of NITR amplicons identify known alleles and reveal additional alleles within the nitr1, nitr2, nitr3, and nitr5 families, reflecting the recent evolution of this gene family.

Hematopoietic cell development in the zebrafish embryo.

PURPOSE OF REVIEW: A wealth of new experimental evidence has been published over the past year that has helped refine our models of blood cell development. We will review this information, discuss the current models of hematopoietic ontogeny and provide perspective on current and future research directions, with an emphasis on how studies in the zebrafish are helping us better understand how hematopoietic stem cells are formed in the vertebrate embryo. RECENT FINDINGS: Several important studies have been published recently addressing the embryonic development of hematopoietic stem cells. These studies have helped clarify several controversial topics in developmental hematopoiesis, including the concepts of the hemangioblast and hemogenic endothelium. In particular, the postulate that hematopoietic stem cells arise through hemogenic endothelial intermediates has been greatly strengthened by a collection of convincing publications reviewed below. SUMMARY: A precise understanding of how hematopoietic stem cells are patterned during development has important implications for both developmental biology and regenerative medicine. Since hematopoietic stem cells are the only hematopoietic cells capable of lifelong, multilineage blood cell production, understanding the stepwise, molecular processes of their instruction from mesoderm is key to replicating these events in vitro from pluripotent embryonic stem cells.

The macrophage-expressed gene (mpeg) 1 identifies a subpopulation of B cells in the adult zebrafish.

The mononuclear phagocytic system consists of many cells, in particular macrophages, scattered throughout the body. However, there is increasing evidence for the heterogeneity of tissue-resident macrophages, leading to a pressing need for new tools to discriminate mononuclear phagocytic system subsets from other hematopoietic lineages. Macrophage-expressed gene (Mpeg)1.1 is an evolutionary conserved gene encoding perforin-2, a pore-forming protein associated with host defense against pathogens. Zebrafish mpeg1.1:GFP and mpeg1.1:mCherry reporters were originally established to specifically label macrophages. Since then more than 100 peer-reviewed publications have made use of mpeg1.1-driven transgenics for in vivo studies, providing new insights into key aspects of macrophage ontogeny, activation, and function. Whereas the macrophage-specific expression pattern of the mpeg1.1 promoter has been firmly established in the zebrafish embryo, it is currently not known whether this specificity is maintained through adulthood. Here we report direct evidence that beside macrophages, a subpopulation of B-lymphocytes is marked by mpeg1.1 reporters in most adult zebrafish organs. These mpeg1.1+ lymphoid cells endogenously express mpeg1.1 and can be separated from mpeg1.1+ macrophages by virtue of their light-scatter characteristics using FACS. Remarkably, our analyses also revealed that B-lymphocytes, rather than mononuclear phagocytes, constitute the main mpeg1.1-positive population in irf8null myeloid-defective mutants, which were previously reported to recover tissue-resident macrophages in adulthood. One notable exception is skin macrophages, whose development and maintenance appear to be independent from irf8, similar to mammals. Collectively, our findings demonstrate that irf8 functions in myelopoiesis are evolutionary conserved and highlight the need for alternative macrophage-specific markers to study the mononuclear phagocytic system in adult zebrafish.

Separation of splenic red and white pulp occurs before birth in a LTalphabeta-independent manner.

For the formation of lymph nodes and Peyer's patches, lymphoid tissue inducer (LTi) cells are crucial in triggering stromal cells to recruit and retain hematopoietic cells. Although LTi cells have been observed in fetal spleen, not much is known about fetal spleen development and the role of LTi cells in this process. Here, we show that LTi cells collect in a periarteriolar manner in fetal spleen at the periphery of the white pulp anlagen. Expression of the homeostatic chemokines can be detected in stromal and endothelial cells, suggesting that LTi cells are attracted by these chemokines. As lymphotoxin (LT)alpha1beta2 can be detected on B cells but not LTi cells in neonatal spleen, starting at 4 days after birth, the earliest formation of the white pulp in fetal spleen occurs in a LTalpha1beta2-independent manner. The postnatal development of the splenic white pulp, involving the influx of T cells, depends on LTalpha1beta2 expressed by B cells.

How HSCs Colonize and Expand in the Fetal Niche of the Vertebrate Embryo: An Evolutionary Perspective.

Rare hematopoietic stem cells (HSCs) can self-renew, establish the entire blood system and represent the basis of regenerative medicine applied to hematological disorders. Clinical use of HSCs is however limited by their inefficient expansion ex vivo, creating a need to further understand HSC expansion in vivo. After embryonic HSCs are born from the hemogenic endothelium, they migrate to the embryonic/fetal niche, where the future adult HSC pool is established by considerable expansion. This takes place at different anatomical sites and is controlled by numerous signals. HSCs then migrate to their adult niche, where they are maintained throughout adulthood. Exactly how HSC expansion is controlled during embryogenesis remains to be characterized and is an important step to improve the therapeutic use of HSCs. We will review the current knowledge of HSC expansion in the different fetal niches across several model organisms and highlight possible clinical applications.