Zebrafish Early Macrophages Colonize Cephalic Mesenchyme and Developing Brain, Retina, and Epidermis through a M-CSF Receptor-Dependent Invasive Process

AbstractThe origin of resident (noninflammatory) macrophages in vertebrate tissues is still poorly understood. In the zebrafish embryo, we recently described a specific lineage of early macrophages that differentiate in the yolk sac before the onset of blood circulation. We now show that these early macrophages spread in the whole cephalic mesenchyme, and from there invade epithelial tissues: epidermis, retina, and brain—especially the optic tectum. In the panther mutant, which lacks a functional fms (M-CSF receptor) gene, early macrophages differentiate and behave apparently normally in the yolk sac, but then fail to invade embryonic tissues. Our video recordings then document for the first time the behavior of macrophages in the invaded tissues, revealing the striking propensity of early macrophages in epidermis and brain to wander restlessly among epithelial cells. This unexpected behavior suggests that tissue macrophages may be constantly “patrolling” for immune and possibly also developmental and trophic surveillance. At 60 h post-fertilization, all macrophages in the brain and retina undergo a specific phenotypic transformation, into “early (amoeboid) microglia”: they become more highly endocytic, they down-regulate the L-plastin gene, and abruptly start expressing high levels of apolipoprotein E, a well-known neurotrophic lipid carrier

A large new subset of TRIM genes highly diversified by duplication and positive selection in teleost fish

<p>Abstract</p> <p>Background</p> <p>In mammals, the members of the tripartite motif (TRIM) protein family are involved in various cellular processes including innate immunity against viral infection. Viruses exert strong selective pressures on the defense system. Accordingly, antiviral TRIMs have diversified highly through gene expansion, positive selection and alternative splicing. Characterizing immune TRIMs in other vertebrates may enlighten their complex evolution.</p> <p>Results</p> <p>We describe here a large new subfamily of TRIMs in teleosts, called finTRIMs, identified in rainbow trout as virus-induced transcripts. FinTRIMs are formed of nearly identical RING/B-box regions and C-termini of variable length; the long variants include a B30.2 domain. The zebrafish genome harbors a striking diversity of finTRIMs, with 84 genes distributed in clusters on different chromosomes. A phylogenetic analysis revealed different subsets suggesting lineage-specific diversification events. Accordingly, the number of <it>fintrim </it>genes varies greatly among fish species. Conserved syntenies were observed only for the oldest <it>fintrims</it>. The closest mammalian relatives are <it>trim16 </it>and <it>trim25</it>, but they are not true orthologs. The B30.2 domain of zebrafish finTRIMs evolved under strong positive selection. The positions under positive selection are remarkably congruent in finTRIMs and in mammalian antiviral TRIM5α, concentrated within a viral recognition motif in mammals. The B30.2 domains most closely related to finTRIM are found among NOD-like receptors (NLR), indicating that the evolution of TRIMs and NLRs was intertwined by exon shuffling.</p> <p>Conclusion</p> <p>The diversity, evolution, and features of finTRIMs suggest an important role in fish innate immunity; this would make them the first TRIMs involved in immunity identified outside mammals.</p

Whole-Body Analysis of a Viral Infection: Vascular Endothelium is a Primary Target of Infectious Hematopoietic Necrosis Virus in Zebrafish Larvae

The progression of viral infections is notoriously difficult to follow in whole organisms. The small, transparent zebrafish larva constitutes a valuable system to study how pathogens spread. We describe here the course of infection of zebrafish early larvae with a heat-adapted variant of the Infectious Hematopoietic Necrosis Virus (IHNV), a rhabdovirus that represents an important threat to the salmonid culture industry. When incubated at 24°C, a permissive temperature for virus replication, larvae infected by intravenous injection died within three to four days. Macroscopic signs of infection followed a highly predictable course, with a slowdown then arrest of blood flow despite continuing heartbeat, followed by a loss of reactivity to touch and ultimately by death. Using whole-mount in situ hybridization, patterns of infection were imaged in whole larvae. The first infected cells were detectable as early as 6 hours post infection, and a steady increase in infected cell number and staining intensity occurred with time. Venous endothelium appeared as a primary target of infection, as could be confirmed in fli1:GFP transgenic larvae by live imaging and immunohistochemistry. Disruption of the first vessels took place before arrest of blood circulation, and hemorrhages could be observed in various places. Our data suggest that infection spread from the damaged vessels to underlying tissue. By shifting infected fish to a temperature of 28°C that is non-permissive for viral propagation, it was possible to establish when virus-generated damage became irreversible. This stage was reached many hours before any detectable induction of the host response. Zebrafish larvae infected with IHNV constitute a vertebrate model of an hemorrhagic viral disease. This tractable system will allow the in vivo dissection of host-virus interactions at the whole organism scale, a feature unrivalled by other vertebrate models

Phosphatidylinositol-3 kinase signaling controls survival and stemness of hematopoietic stem and progenitor cells

Hematopoietic stem and progenitor cells (HSPCs) are multipotent cells giving rise to all blood lineages during life. HSPCs emerge from the ventral wall of the dorsal aorta (VDA) during a specific timespan in embryonic development through endothelial hematopoietic transition (EHT). We investigated the ontogeny of HSPCs in mutant zebrafish embryos lacking functional pten, an important tumor suppressor with a central role in cell signaling. Through in vivo live imaging, we discovered that in pten mutant embryos a proportion of the HSPCs died upon emergence from the VDA, an effect rescued by inhibition of phosphatidylinositol-3 kinase (PI3K). Surprisingly, inhibition of PI3K in wild-type embryos also induced HSPC death. Surviving HSPCs colonized the caudal hematopoietic tissue (CHT) normally and committed to all blood lineages. Single-cell RNA sequencing indicated that inhibition of PI3K enhanced survival of multipotent progenitors, whereas the number of HSPCs with more stem-like properties was reduced. At the end of the definitive wave, loss of Pten caused a shift to more restricted progenitors at the expense of HSPCs. We conclude that PI3K signaling tightly controls HSPCs survival and both up- and downregulation of PI3K signaling reduces stemness of HSPCs

The zebrafish as a new model for the in vivo study of Shigella flexneri interaction with phagocytes and bacterial autophagy.

Autophagy, an ancient and highly conserved intracellular degradation process, is viewed as a critical component of innate immunity because of its ability to deliver cytosolic bacteria to the lysosome. However, the role of bacterial autophagy in vivo remains poorly understood. The zebrafish (Danio rerio) has emerged as a vertebrate model for the study of infections because it is optically accessible at the larval stages when the innate immune system is already functional. Here, we have characterized the susceptibility of zebrafish larvae to Shigella flexneri, a paradigm for bacterial autophagy, and have used this model to study Shigella-phagocyte interactions in vivo. Depending on the dose, S. flexneri injected in zebrafish larvae were either cleared in a few days or resulted in a progressive and ultimately fatal infection. Using high resolution live imaging, we found that S. flexneri were rapidly engulfed by macrophages and neutrophils; moreover we discovered a scavenger role for neutrophils in eliminating infected dead macrophages and non-immune cell types that failed to control Shigella infection. We observed that intracellular S. flexneri could escape to the cytosol, induce septin caging and be targeted to autophagy in vivo. Depletion of p62 (sequestosome 1 or SQSTM1), an adaptor protein critical for bacterial autophagy in vitro, significantly increased bacterial burden and host susceptibility to infection. These results show the zebrafish larva as a new model for the study of S. flexneri interaction with phagocytes, and the manipulation of autophagy for anti-bacterial therapy in vivo

Recherche et caractérisation de mutants de Danio rerio affectés dans le développement des macrophages primitifs et de la microglie

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Caractérisation génétique et phénotypique de mutants de Danio rerio dépourvus d'une population spécifique de macrophages primitifs (la micrologie du Système Nerveux Central)

Chez l ensemble des vertébrés, les macrophages sont les premières cellules du système immunitaire à être différenciées au cours du développement embryonnaire. Ces cellules myéloïdes phagocytaires, générées au niveau du sac vitellin, se dispersent dans les tissus, pour y accomplir leurs fonctions de protection face aux infections, et d élimination des corps apoptotiques. Ceux de ces macrophages primitifs qui colonisent le Système Nerveux Central (SNC) s y différencient en microglie, par l acquisition d une morphologie très ramifiée et une diminution du comportement migratoire. L objectif de ma thèse était de découvrir des gènes impliqués dans ces différentes étapes de l établissement des populations de macrophages tissulaires : migration, invasion, survie, maintien et/ou renouvellement. Pour cela, j ai étudié 5 mutants isolés par le laboratoire comme dépourvus de microglie à 4 jours. La caractérisation du phénotype m a permis d établir que 3 des mutants présentent une absence de colonisation du SNC par les macrophages primitifs ; deux d entre eux appartiennent au même groupe de complémentation (grenadineNT080 et grenadineNC081) et ne présentent pas d épistasie avec le 3e (vermillon). Les macrophages primitifs d un des mutants (brique), dont la microglie, sont incapables de digérer le contenu de leurs phagosomes. Enfin, deux mutants présentent un défaut de survie des macrophages primitifs dans les tissus. Dans le cas de cerise, la survie de ces cellules n est affectée que dans le SNC, tandis que chez moonshine, ils meurent simultanément dans l ensemble des tissus de l embryon. Les gènes sont identifiés pour les mutants cerise (slc7a7) et moonshine (Trim33), et sont connus pour jouer un rôle, respectivement dans le fonctionnement des macrophages chez les mammifères, et l hématopoïèse chez tous les vertébrés. Les autres gènes devraient être identifiés sous peu et permettre d attribuer des acteurs moléculaires supplémentaires à ces différentes étapes de colonisation, fonction et maintien des populations de macrophages tissulaires, et d avancer dans leur compréhension. Ces mutants seront d excellents outils pour appréhender le rôle de la microglie et le développement du SNC en son absence (cerise, grenadine et vermillon) ainsi que l évolution des infections en l absence de macrophages (moonshine) ou lorsque leurs capacités de digestion sont altérées (brique). Ces gènes, comme c est le cas pour slc7a7 affecté chez cerise, peuvent être impliqués dans des pathologies humaines, faisant de ces mutants des modèles d étude de ces maladiesIn all vertebrate embryos, the primitive macrophages are the first immune cells to differentiate in the yolk sac. These professional phagocytes quickly spread in all the embryonic tissues, including the Central Nervous System (CNS), where they differentiate into ramified microglia. In order to identify new genes involved in this establishment of tissue-specific macrophages populations, I studied 5 mutants identified by the lab as lacking microglia at 4 days post-fertilization (dpf). Phenotypic characterization permitted to establish that, in 3 of the mutants, macrophages are unable to invade the CNS. The 3 mutants are grenadineNT080 and grenadineNC081 (that belong to the same complementation group) and vermillon. In the mutant brique, all primitive macrophages show a defect in digesting their phagosomes. Finally, macrophage survival is affected in both mutants cerise and moonshine. In the cerise mutant, macrophages die specifically inside the SNC, whereas in moonshine, their survival is affected in all embryonic tissues. The mutated genes have been identified for cerise (slc7a7) and moonshine (Trim33). Very interestingly, these two genes are already known to play a role in macrophage biology and hematopoiesis respectively. The other genes should be identified soon, leading to a better understanding of the establishment and maintaining of tissue-specific macrophage populations. These mutants will be valuable tools to assess the role of microglia, and the development of the brain in absence of this population (cerise, vermillon and grenadine), as well as the course of infections when professional phagocytes are absent (moonshine) or not fully functional (brique). Their further analysis may lead to establish parallels with some human pathology, as it did for cerise, and thus make some of these mutants precious living models for these diseasesPARIS-BIUSJ-Biologie recherche (751052107) / SudocSudocFranceF