Characterization of the mononuclear phagocyte system in zebrafish

Abstract

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 antigenpresenting 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 doubletransgenic 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. (Blood. 2011;117(26):7126-7135) IntroductionIn recent years, the zebrafish (Danio rerio) has proven to be a unique vertebrate model for the study of hematopoiesis. 1 The use of the zebrafish to study the ontogeny of leukocyte subsets, 2 immune cell migration, 3,4 and host-pathogen interactions 5 has provided new insights into our understanding of innate immunity in the developing vertebrate embryo. A major focus of previous studies was on the neutrophil response, because several transgenic reporter lines have been generated that mark this granulocyte subset. Whereas neutrophils generally constitute the first line of defense against invading pathogens, the role of other immune cell subsets in the innate immune response has received less attention. In addition to neutrophils, macrophages are key in the response to pathogen challenge. In the zebrafish embryo, primitive macrophages have been demonstrated to be capable of clearing injected bacteria by phagocytosis. 6 However, the absence of markers specific to macrophages has limited the study of this myeloid cell subset in the zebrafish.The mononuclear phagocyte system (MPS) comprises monocytes, tissue macrophages, and dendritic cells (DCs), as well as their lineage-committed progenitors. 7 The primary function of mature MPS cells is the clearance of pathogens by phagocytosis. This activity is crucial during immune challenge to clear invasive pathogens. Mononuclear phagocytes also play an important role in the removal of apoptotic cell corpses, especially during embryonic development. In mice, embryonic macrop...

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

“…Whereas the endogenous cd45 gene is expressed in all zebrafish leukocytes, we previously showed that a cd45:DsRed transgene marks T cells but not B cells. 9 Flow cytometric analysis of lymphocytes and precursors from the kidneys of cd45:DsRed;blimp1:GFP fish revealed a small CD45 2 Blimp1 1 population, as well as larger populations of Figure 6B), in agreement with results for mammalian plasma B cells. 33 Further qRT-PCR analysis of this population revealed that these cells also expressed other markers of plasma B cells, 41 including cd40 and the transcription factor X-box binding protein 1 (xbp1) and interferon regulatory factor 4 (irf4) ( Figure 6B).…”

“…We note that this is because of a unique feature of this CD45-transgenic line whereby DsRed is expressed in all leukocytes except B cells. 9 Thus, flow cytometric analysis of IgM1:eGFP;cd45:DsRed animals yields GFP …”

“…9,23 Flow cytometry was performed with a fluorescence-activated cell sorter (FACS) AriaI or FACSCanto flow cytometer (Becton Dickinson). Cytospins and May-Grunwald/Giemsa stains were performed as described.…”

“…4 Several transgenic reporter lines, in which cell-specific enhancers drive the expression of fluorescent proteins, have been created to label specific blood cells, including T cells, erythrocytes, eosinophils, neutrophils, macrophages, and antigenpresenting cells. [5][6][7][8][9] By combining fluorescent transgenesis with advanced imaging techniques, zebrafish offer unique advantages over other vertebrate models for visualizing the behavior of immune cells in living animals. Currently, no transgenic reporter lines exist that specifically label B lymphocytes in zebrafish, or in any other nonmammalian animal model.…”

Vertebrate Epidermal Cells Are Broad-Specificity Phagocytes That Clear Sensory Axon Debris

“…A previous study found that cells with the stellate morphology characteristic of Langerhans cells, blood-derived professional phagocytes resident to vertebrate skin (Carrillo-Farga et al, 1990;Lugo-Villarino et al, 2010), populate the zebrafish epidermis by 12 d postfertilization (dpf; Wittamer et al, 2011). To determine whether epidermal cells still participate in clearance when these other phagocytic cells are present, we repeated these experiments at a later stage.…”