Summary Hematopoietic stem cells (HSCs) underlie the production of blood and immune cells for the lifetime of an organism. In vertebrate embryos, HSCs arise from the unique transdifferentiation of hemogenic endothelium comprising the floor of the dorsal aorta during a brief developmental window. To date, this process has not been replicated in vitro from pluripotent precursors, partly because the full complement of required signaling inputs remains to be determined. Here, we show that TNFR2 via TNFα activates the Notch and NF-κB signaling pathways to establish HSC fate, indicating a requirement for inflammatory signaling in HSC generation. We determine that primitive neutrophils are the major source of TNFα, assigning a role for transient innate immune cells in establishing the HSC program. These results demonstrate that proinflammatory signaling, in the absence of infection, is utilized by the developing embryo to generate the lineal precursors of the adult hematopoietic system.

The adult blood system is established by hematopoietic stem cells (HSCs), which arise during development from an endothelial-tohematopoietic 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.

Calreticulin is a calcium-binding chaperone that has several functions in the immune response. In the endoplasmic reticulum (ER), calreticulin facilitates the folding of major histocompatibility complex (MHC) class I molecules and their assembly factor tapasin, thereby influencing antigen presentation to cytotoxic T cells. Although calreticulin is normally ER-resident, it is found at the cell surface of living cancer cells and dying cells. Here, calreticulin promotes cellular phagocytic uptake. In tumor vaccine models, drugs that induce cell-surface calreticulin confer enhanced tumor protection in an extracellular calreticulin-dependent manner. Much remains to be understood about the roles of calreticulin in these distinct functions. Further investigations are important towards advancing basic knowledge of glycoprotein folding pathways, and towards developing new cancer therapeutic strategies.

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Major histocompatibility complex (MHC) class I molecules are ligands for T-cell receptors of CD8؉ T cells and inhibitory S1A), using the crystal structure of the closely related protein, calnexin (2). Calreticulin is thought to contain three structural domains. The first is a large globular domain comprising the Nand C-terminal regions of the protein that form a ␤-stranded sandwich and a C-terminal helix (supplemental Fig. S1A, orange, light blue, and green). The glycan binding site is located within this domain (supplemental Fig. S1B). A second hookshaped P-domain forms a ␤-stranded hairpin structure inserted in the middle of the globular domain (supplemental Fig. S1A, dark blue). The tip of this domain forms a binding site for ERp57 (3), an ER oxidoreductase that works cooperatively with calreticulin and calnexin in glycoprotein folding (4). A third C-terminal domain, rich in acidic amino acids, is functional as a low affinity/high capacity calcium coordination site (1). This region is not present in the lumenal domain of calnexin.In vitro studies have shown that calreticulin can bind to misfolded non-glycosylated polypeptides and suppress their irreversible aggregation (5). This activity is induced by various conditions associated with ER stress, including calcium depletion and heat shock (6). These conditions also induce calreticulin oligomerization (6, 7). Much remains to be understood about the one or more binding sites on calreticulin that are used to suppress substrate aggregation, as well as the relevance of this activity to calreticulin-mediated protein folding under physiological non-stress conditions.Calreticulin is a key player in the MHC class I assembly pathway (8). The MHC class I-dedicated assembly factors, transporter associated with antigen processing (TAP) and tapasin, as well as the generic ER-folding factors ERp57 and calreticulin, form a large complex with MHC class I molecules, collectively called the PLC. TAP provides a major source of peptides for MHC class I molecules, whereas tapasin, ERp57, and calreticulin facilitate assembly of MHC class I molecules with peptides (reviewed in Ref. 9). Calreticulin is a component of the PLC, and calreticulin-deficient cells express reduced cell surface MHC class I molecules (8). The mechanisms by which calreticulin contributes to enhanced MHC class I assembly are not well understood.Early studies with glycosylation inhibitors, MHC class I mutants, and in vitro binding analyses suggested that glycanbased interactions with MHC class I molecules recruit calreti-* This work was supported, in whole or in part, by National Institutes of Health Grant AI066131 (to M. R.) and by a diversity supplement to National Institutes of Health Grant AI066131 (awarded to N. D.). □ S The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S3 and

The assembly of major histocompatibility complex (MHC) class I molecules with peptides is orchestrated by several assembly factors including the transporter associated with antigen processing (TAP) and tapasin, the endoplasmic reticulum (ER) oxido-reductases ERp57 and protein disulfide isomerase (PDI), the lectin chaperones calnexin and calreticulin, and the ER aminopeptidase (ERAAP). Typically, MHC class I molecules present endogenous antigens to cytotoxic T lymphocytes (CTLs). However, the initiation of CD8 + T-cell responses against many pathogens and tumors also requires the presentation of exogenous antigens by MHC class I molecules. We discuss recent developments relating to interactions and mechanisms of function of the various assembly factors and pathways by which exogenous antigens access MHC class I molecules.

Streptococcus agalactiae (Group B Streptococcus, GBS) is an encapsulated, Gram-positive bacterium that is a leading cause of neonatal pneumonia, sepsis and meningitis, and an emerging aquaculture pathogen. The zebrafish (Danio rerio) is a genetically tractable model vertebrate that has been used to analyze the pathogenesis of both aquatic and human bacterial pathogens. We have developed a larval zebrafish model of GBS infection to study bacterial and host factors that contribute to disease progression. GBS infection resulted in dose dependent larval death, and GBS serotype III, ST-17 strain was observed as the most virulent. Virulence was dependent on the presence of the GBS capsule, surface anchored lipoteichoic acid (LTA) and toxin production, as infection with GBS mutants lacking these factors resulted in little to no mortality. Additionally, interleukin-1β il1b and CXCL-8 (cxcl8a) were significantly induced following GBS infection compared to controls. We also visualized GBS outside the brain vasculature, suggesting GBS penetration into the brain during the course of infection. Our data demonstrate that zebrafish larvae are a valuable model organism to study GBS pathogenesis.

Antigen presentation is a critical step in the activation of naïve T lymphocytes. In mammals, dendritic cells (DCs), macrophages, and B lymphocytes can all function as antigen presenting cells (APCs). Although APCs have been identified in zebrafish, it is unclear if they fulfill similar roles in the initiation of adaptive immunity. Here we review the characterization of zebrafish macrophages, DCs, and B cells and evidence of their function as true APCs. Finally, we discuss the conservation of APC activity in vertebrates and the use of zebrafish to provide a new perspective on the evolution of these functions.