David L. Stachura

Definitive hematopoiesis initiates through a committed erythromyeloid progenitor in the zebrafish embryo

Shifting sites of blood cell production during development is common across widely divergent phyla. In zebrafish, like other vertebrates, hematopoietic development has been roughly divided into two waves, termed primitive and definitive. Primitive hematopoiesis is characterized by the generation of embryonic erythrocytes in the intermediate cell mass and a distinct population of macrophages that arises from cephalic mesoderm. Based on previous gene expression studies, definitive hematopoiesis has been suggested to begin with the generation of presumptive hematopoietic stem cells (HSCs) along the dorsal aorta that express c-myb and runx1. Here we show, using a combination of gene expression analyses, prospective isolation approaches, transplantation, and in vivo lineage-tracing experiments, that definitive hematopoiesis initiates through committed erythromyeloid progenitors (EMPs) in the posterior blood island (PBI) that arise independently of HSCs. EMPs isolated by coexpression of fluorescent transgenes driven by the lmo2 and gata1 promoters exhibit an immature, blastic morphology and express only erythroid and myeloid genes. Transplanted EMPs home to the PBI, show limited proliferative potential, and do not seed subsequent hematopoietic sites such as the thymus or pronephros. In vivo fate-mapping studies similarly demonstrate that EMPs possess only transient proliferative potential, with differentiated progeny remaining largely within caudal hematopoietic tissue. Additional fate mapping of mesodermal derivatives in mid-somitogenesis embryos suggests that EMPs are born directly in the PBI. These studies provide phenotypic and functional analyses of the first hematopoietic progenitors in the zebrafish embryo and demonstrate that definitive hematopoiesis proceeds through two distinct waves during embryonic development.

Identification of dendritic antigen-presenting cells in the zebrafish

In mammals, dendritic cells (DCs) form the key link between the innate and adaptive immune systems. DCs act as immune sentries in various tissues and, upon encountering pathogen, engulf and traffic foreign antigen to secondary lymphoid tissues, stimulating antigen-specific T lymphocytes. Although DCs are of fundamental importance in orchestrating the mammalian immune response, their presence and function in nonmammalian vertebrates is largely unknown. Because teleosts possess one of the earliest recognizable adaptive immune systems, we sought to identify antigen-presenting cells (APCs) in the zebrafish to better understand the potential origins of DCs and their evolutionary relationship to lymphocytes. Here we present the identification and characterization of a zebrafish APC subset strongly resembling mammalian DCs. Rare DCs are present in various adult tissues, and can be enriched by their affinity for the lectin peanut agglutinin (PNA). We show that PNAhi myeloid cells possess the classical morphological features of mammalian DCs as revealed by histochemical and ultrastructural analyses, phagocytose-labeled bacterial preparations in vivo, and exhibit expression of genes associated with DC function and antigen presentation, including il12, MHC class II invariant chain iclp1, and csf1r. Importantly, we show that PNAhi cells can activate T lymphocytes in an antigen-dependent manner. Together, these studies suggest that the cellular constituents responsible for antigen presentation are remarkably conserved from teleosts to mammals, and indicate that the zebrafish may serve as a unique model to study the origin of APC subsets and their evolutionary role as the link between the innate and adaptive immune systems.

Eosinophils in the zebrafish: prospective isolation, characterization, and eosinophilia induction by helminth determinants

Eosinophils are granulocytic leukocytes implicated in numerous aspects of immunity and disease. The precise functions of eosinophils, however, remain enigmatic. Alternative models to study eosinophil biology may thus yield novel insights into their function. Eosinophilic cells have been observed in zebrafish but have not been thoroughly characterized. We used a gata2:eGFP transgenic animal to enable prospective isolation and characterization of zebrafish eosinophils, and demonstrate that all gata2(hi) cells in adult hematopoietic tissues are eosinophils. Although eosinophils are rare in most organs, they are readily isolated from whole kidney marrow and abundant within the peritoneal cavity. Molecular analyses demonstrate that zebrafish eosinophils express genes important for the activities of mammalian eosinophils. In addition, gata2(hi) cells degranulate in response to helminth extract. Chronic exposure to helminth- related allergens resulted in profound eosinophilia, demonstrating that eosinophil responses to allergens have been conserved over evolution. Importantly, infection of adult zebrafish with Pseudocapillaria tomentosa, a natural nematode pathogen of teleosts, caused marked increases in eosinophil number within the intestine. Together, these observations support a conserved role for eosinophils in the response to helminth antigens or infection and provide a new model to better understand how parasitic worms activate, co-opt, or evade the vertebrate immune response.

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.

Cellular Dissection of Zebrafish Hematopoiesis

The zebrafish is an excellent model system to study vertebrate blood cell development due to a highly conserved hematopoietic system, optical transparency, and amenability to both forward and reverse genetic approaches. The development of functional assays to analyze the biology of hematopoietic mutants and diseased animals remains a work in progress. Here we discuss recent advances in zebrafish hematology, prospective isolation techniques, cellular transplantation, and culture-based assays that now provide more rigorous tests of hematopoietic stem and progenitor cell function. Together with the proven strengths of the zebrafish, the development and refinement of these assays further enable efforts to better understand the development and evolution of the vertebrate hematopoietic system.

Discrete Notch signaling requirements in the specification of hematopoietic stem cells

Hematopoietic stem cells (HSCs) require multiple molecular inputs for proper specification, including activity of the Notch signaling pathway. A requirement for the Notch1 and dispensability of the Notch2 receptor has been demonstrated in mice, but the role of the remaining Notch receptors has not been investigated. Here, we demonstrate that three of the four Notch receptors are independently required for the specification of HSCs in the zebrafish. The orthologues of the murine Notch1 receptor, Notch1a and Notch1b, are each required intrinsically to fate HSCs, just prior to their emergence from aortic hemogenic endothelium. By contrast, the Notch3 receptor is required earlier within the developing somite to regulate HSC emergence in a non‐cell‐autonomous manner. Epistatic analyses demonstrate that Notch3 function lies downstream of Wnt16, which is required for HSC specification through its regulation of two Notch ligands, dlc and dld. Collectively, these findings demonstrate for the first time that multiple Notch signaling inputs are required to specify HSCs and that Notch3 performs a novel role within the somite to regulate the neighboring precursors of hemogenic endothelium.

Zebrafish kidney stromal cell lines support multilineage hematopoiesis

Studies of zebrafish hematopoiesis have been largely performed using mutagenesis approaches and retrospective analyses based upon gene expression patterns in whole embryos. We previously developed transplantation assays to test the repopulation potentials of candidate hematopoietic progenitor cells. We have been impaired, however, in determining cellular differentiation potentials by a lack of short-term functional assays. To enable more precise analyses of hematopoietic progenitor cells, we have created zebrafish kidney stromal (ZKS) cell lines. Culture of adult whole kidney marrow with ZKS cells results in the maintenance and expansion of hematopoietic precursor cells. Hematopoietic growth is dependent upon ZKS cells, and we show that ZKS cells express many growth factors and ligands previously demonstrated to be important in maintaining mammalian hematopoietic cells. In the absence of exogenous growth factors, ZKS cells maintain early hematopoietic precursors and support differentiation of lymphoid and myeloid cells. With the addition of zebrafish erythropoietin, ZKS cells also support the differentiation of erythroid precursors. These conditions have enabled the ability to ascertain more precisely the points at which hematopoietic mutants are defective. The development of robust in vitro assays now provide the means to track defined, functional outcomes for prospectively isolated blood cell subsets in the zebrafish.

Gata2b is a restricted early regulator of hemogenic endothelium in the zebrafish embryo

The adult blood system is established by hematopoietic stem cells (HSCs), which arise during development from an endothelial-to-hematopoietic transition of cells comprising the floor of the dorsal aorta. Expression of aortic runx1 has served as an early marker of HSC commitment in the zebrafish embryo, but recent studies have suggested that HSC specification begins during the convergence of posterior lateral plate mesoderm (PLM), well before aorta formation and runx1 transcription. Further understanding of the earliest stages of HSC specification necessitates an earlier marker of hemogenic endothelium. Studies in mice have suggested that GATA2 might function at early stages within hemogenic endothelium. Two orthologs of Gata2 exist in zebrafish: gata2a and gata2b. Here, we report that gata2b expression initiates during the convergence of PLM, becoming restricted to emerging HSCs. We observe Notch-dependent gata2b expression within the hemogenic subcompartment of the dorsal aorta that is in turn required to initiate runx1 expression. Our results indicate that Gata2b functions within hemogenic endothelium from an early stage, whereas Gata2a functions more broadly throughout the vascular system. Highlighted article: Gata2b marks a distinct population of embryonic endothelial cells that gives rise to hematopoietic stem cells and is required for the hemogenic potential of these cells.

The zebrafish granulocyte colony-stimulating factors (Gcsfs): 2 paralogous cytokines and their roles in hematopoietic development and maintenance

Granulocyte colony-stimulating factor (Gcsf) drives the proliferation and differentiation of granulocytes, monocytes, and macrophages (mφs) from hematopoietic stem and progenitor cells (HSPCs). Analysis of the zebrafish genome indicates the presence of 2 Gcsf ligands, likely resulting from a duplication event in teleost evolution. Although Gcsfa and Gcsfb share low sequence conservation, they share significant similarity in their predicted ligand/receptor interaction sites and structure. Each ligand displays differential temporal expression patterns during embryogenesis and spatial expression patterns in adult animals. To determine the functions of each ligand, we performed loss- and gain-of-function experiments. Both ligands signal through the Gcsf receptor to expand primitive neutrophils and mφs, as well as definitive granulocytes. To further address their functions, we generated recombinant versions and tested them in clonal progenitor assays. These sensitive in vitro techniques indicated similar functional attributes in supporting HSPC growth and differentiation. Finally, in addition to supporting myeloid differentiation, zebrafish Gcsf is required for the specification and proliferation of hematopoietic stem cells, suggesting that Gcsf represents an ancestral cytokine responsible for the broad support of HSPCs. These findings may inform how hematopoietic cytokines evolved following the diversification of teleosts and mammals from a common ancestor.

FGF signalling specifies haematopoietic stem cells through its regulation of somitic Notch signalling

Hematopoietic stem cells (HSCs) derive from hemogenic endothelial cells of the primitive dorsal aorta (DA) during vertebrate embryogenesis. The molecular mechanisms governing this unique endothelial to hematopoietic transition remain unclear. Here, we demonstrate a novel requirement for fibroblast growth factor (FGF) signaling in HSC emergence. This requirement is non-cell-autonomous, and acts within the somite to bridge the Wnt and Notch signaling pathways. We previously demonstrated that Wnt16 regulates the somitic expression of two Notch ligands, deltaC (dlc) and deltaD (dld), whose combined function is required for HSC fate. How Wnt16 connects to Notch function has remained an open question. Our current studies demonstrate that FGF signaling, via FGF receptor 4 (Fgfr4), mediates a signal transduction pathway between Wnt16 and Dlc, but not Dld, to regulate HSC specification. Our findings demonstrate that FGF signaling acts as a key molecular relay within the developmental HSC niche to instruct HSC fate.