Karima Kissa

Blood stem cells emerge from aortic endothelium by a novel type of cell transition

The ontogeny of haematopoietic stem cells (HSCs) during embryonic development is still highly debated, especially their possible lineage relationship to vascular endothelial cells. The first anatomical site from which cells with long-term HSC potential have been isolated is the aorta-gonad-mesonephros (AGM), more specifically the vicinity of the dorsal aortic floor. But although some authors have presented evidence that HSCs may arise directly from the aortic floor into the dorsal aortic lumen, others support the notion that HSCs first emerge within the underlying mesenchyme. Here we show by non-invasive, high-resolution imaging of live zebrafish embryos, that HSCs emerge directly from the aortic floor, through a stereotyped process that does not involve cell division but a strong bending then egress of single endothelial cells from the aortic ventral wall into the sub-aortic space, and their concomitant transformation into haematopoietic cells. The process is polarized not only in the dorso-ventral but also in the rostro-caudal versus medio-lateral direction, and depends on Runx1 expression: in Runx1-deficient embryos, the exit events are initially similar, but much rarer, and abort into violent death of the exiting cell. These results demonstrate that the aortic floor is haemogenic and that HSCs emerge from it into the sub-aortic space, not by asymmetric cell division but through a new type of cell behaviour, which we call an endothelial haematopoietic transition.

Preferential transduction of neurons by canine adenovirus vectors and their efficient retrograde transport in vivo

In the central nervous system (CNS), there are innate obstacles to the modification of neurons: their relative low abundance versus glia and oligodendrocytes, the inaccessibility of certain target populations, and the volume one can inject safely. Our aim in this study was to characterize the in vivo efficacy of a novel viral vector derived from a canine adenovirus (CAV‐2). Here we show that CAV‐2 preferentially transduced i) rat olfactory sensory neurons; ii) rodent CNS neurons in vitro and in vivo; and, more clinically relevant, iii) neurons in organotypic slices of human cortical brain. CAV‐2 also showed a high disposition for retrograde axonal transport in vivo. We examined the molecular basis of neuronal targeting by CAV‐2 and suggest that due to CAR (coxsackie adenovirus receptor) expression on neuronal cells—and not oligodendrocytes, glia, myofibers, and nasal epithelial cells—CAV‐2 vectors transduced neurons preferentially in these diverse tissues.

Mycobacterium abscessus cording prevents phagocytosis and promotes abscess formation

Significance Mycobacterium abscessus is the most frequently isolated rapidly growing mycobacterium in human disease and recently has emerged as responsible for severe pulmonary infections in cystic fibrosis patients. However, little is known about the virulence mechanisms of this human pathogen. We adapted the zebrafish embryo as a tractable infection model to study, at a spatiotemporal level, the physiopathology of M. abscessus infection. We describe the high propensity of virulent rough variant M. abscessus to produce serpentine cords in vivo, which are not observed with the less virulent smooth variant. We demonstrate that extracellular cording allows the bacterium to withstand phagocytosis, leading to uncontrolled growth and establishment of an acute and lethal infection, thus constituting a determinant of virulence. Mycobacterium abscessus is a rapidly growing Mycobacterium causing a wide spectrum of clinical syndromes. It now is recognized as a pulmonary pathogen to which cystic fibrosis patients have a particular susceptibility. The M. abscessus rough (R) variant, devoid of cell-surface glycopeptidolipids (GPLs), causes more severe clinical disease than the smooth (S) variant, but the underlying mechanisms of R-variant virulence remain obscure. Exploiting the optical transparency of zebrafish embryos, we observed that the increased virulence of the M. abscessus R variant compared with the S variant correlated with the loss of GPL production. The virulence of the R variant involved the massive production of serpentine cords, absent during S-variant infection, and the cords initiated abscess formation leading to rapid larval death. Cording occurred within the vasculature and was highly pronounced in the central nervous system (CNS). It appears that M. abscessus is transported to the CNS within macrophages. The release of M. abscessus from apoptotic macrophages initiated the formation of cords that grew too large to be phagocytized by macrophages or neutrophils. This study is a description of the crucial role of cording in the in vivo physiopathology of M. abscessus infection and emphasizes cording as a mechanism of immune evasion.

Retrograde trans-synaptic transfer of green fluorescent protein allows the genetic mapping of neuronal circuits in transgenic mice

The function of the nervous system is a consequence of the intricate synaptic connectivity of its neurons. Our understanding of these highly complex networks has profited enormously from methods used over the past two decades that are based on the mechanical injection of tracer molecules into brain regions. We have developed a genetic system for the mapping of synaptic connections during development of the mammalian central nervous system and in the mature brain. It is based on the transsynaptic transfer of green fluorescent protein (GFP) in the brains of mice using a fusion protein with a nontoxic fragment of tetanus toxin (TTC) expressed in defined neurons. These transgenic mice allowed us to visualize neurons, at single-cell resolution, that are in synaptic contact by the detection of GFP in interconnected circuits. Targeted genetic expression with a specific promoter permitted us to transfer GFP to defined subsets of neurons and brain regions. GFP–TTC is coexpressed with a lacZ reporter gene to discriminate neurons that produce the tracer from cells that have acquired it transneuronally. The marker shows selective transfer in the retrograde direction. We have used electron microscopic detection of GFP to define the ultrastructural features of the system. Our work opens up a range of possibilities for brain slice and in vivo studies taking advantage of the fluorescence of GFP. We point the way toward the use of powerful multiphoton technology and set the stage for the transsynaptic transfer of other proteins in the brains of mice.

Real-Time Observation of Listeria monocytogenes-Phagocyte Interactions in Living Zebrafish Larvae†

ABSTRACT The zebrafish, Danio rerio, has become a popular vertebrate model for the study of infections, mainly because of its excellent optical accessibility at the embryonic and larval stages, when the innate immune system is already effective. We have thus tested the susceptibility of zebrafish larvae to the human pathogen Listeria monocytogenes, a gram-positive, facultative, intracellular bacterium that is known to survive and multiply in professional phagocytes and that causes fatal meningitis and abortions. Intravenous injection of early zebrafish larvae resulted in a progressive and ultimately fatal infection. Blood-borne L. monocytogenes bacteria were quickly trapped and engulfed by macrophages, an event that, for the first time, could be captured in vivo and in real time. Granulocytes also participated in the innate immune response. As in mammals, bacteria could escape the macrophage phagosome in a listeriolysin-dependent manner and accessed the cytosol; this event was critical for bacterial virulence, as listeriolysin-deficient bacteria were completely avirulent. Actin comet tails and protrusions were observed, suggesting cell-to-cell spread; these phenomena also played a role in virulence in zebrafish larvae, as actA-deficient bacteria were attenuated. These results demonstrate the relevance of the genetically tractable and optically accessible zebrafish model for the study of L. monocytogenes pathogenesis and particularly for the dissection of its interactions with phagocytes in vivo, a key factor of L. monocytogenes virulence.

Piezo1 plays a role in erythrocyte volume homeostasis

Mechanosensitivity is an inherent property of virtually all cell types, allowing them to sense and respond to physical environmental stimuli. Stretch-activated ion channels represent a class of mechanosensitive proteins which allow cells to respond rapidly to changes in membrane tension; however their identity has remained elusive. The piezo genes have recently been identified as a family of stretch-activated mechanosensitive ion channels. We set out to determine the role of piezo1 during zebrafish development. Here we report that morpholino-mediated knockdown of piezo1 impairs erythrocyte survival without affecting hematopoiesis or differentiation. Our results demonstrate that piezo1 is involved in erythrocyte volume homeostasis, disruption of which results in swelling/lysis of red blood cells and consequent anemia.

In Vivo Neuronal Tracing with GFP-TTC Gene Delivery

The retrograde transport and transynaptic transfer properties of the nontoxic tetanus toxin C-fragment (TTC) can be used to visualize specific neural pathways or to deliver biomolecules in the central nervous system (CNS). Here we tested different delivery techniques to explore the potential use of a new GFP-TTC fusion construct for use as a genetic tracer in vivo. Plasmids encoding GFP-TTC were targeted to brain regions using intracerebral grafted transfected cells or adenoviral transduction. Transport was monitored using GFP fluorescence. We show that following GFP-TTC synthesis in grafted transfected cells, the TTC fragment alone, with no signal peptide, is necessary and sufficient to provide secretion and uptake of the fusion protein into neighboring neurons around the injection site. Using an adenoviral vector to express the fusion protein into brain neurons, we show that transduced neurons can deliver the fusion protein specifically into connected neurons, demonstrating that synaptic transfer in the CNS can be visualized with GFP-TTC.

Identification of polarized macrophage subsets in zebrafish

While the mammalian macrophage phenotypes have been intensively studied in vitro, the dynamic of their phenotypic polarization has never been investigated in live vertebrates. We used the zebrafish as a live model to identify and trail macrophage subtypes. We generated a transgenic line whose macrophages expressing tumour necrosis factor alpha (tnfa), a key feature of classically activated (M1) macrophages, express fluorescent proteins Tg(mpeg1:mCherryF/tnfa:eGFP-F). Using 4D-confocal microscopy, we showed that both aseptic wounding and Escherichia coli inoculation triggered macrophage recruitment, some of which started to express tnfa. RT-qPCR on Fluorescence Activated Cell Sorting (FACS)-sorted tnfa+ and tnfa− macrophages showed that they, respectively, expressed M1 and alternatively activated (M2) mammalian markers. Fate tracing of tnfa+ macrophages during the time-course of inflammation demonstrated that pro-inflammatory macrophages converted into M2-like phenotype during the resolution step. Our results reveal the diversity and plasticity of zebrafish macrophage subsets and underline the similarities with mammalian macrophages proposing a new system to study macrophage functional dynamic. DOI: http://dx.doi.org/10.7554/eLife.07288.001

Primitive macrophages control HSPC mobilization and definitive haematopoiesis

In vertebrates, haematopoietic stem/progenitor cells (HSPCs) first emerge in the aorta-gonad-mesonephros (AGM) before colonizing transitory and subsequently definitive haematopoietic organs allowing haematopoiesis throughout adult life. Here we identify an unexpected primitive macrophage population accumulated in the dorsal mesenteric mesoderm surrounding the dorsal aorta of the human embryo and study its function in the transparent zebrafish embryo. Our study reveals dynamic interactions occurring between the HSPCs and primitive macrophages in the AGM. Specific chemical and inducible genetic depletion of macrophages or inhibition of matrix metalloproteinases (Mmps) leads to an accumulation of HSPCs in the AGM and a decrease in the colonization of haematopoietic organs. Finally, in vivo zymography demonstrates the function of primitive macrophages in extracellular matrix degradation, which allows HSPC migration through the AGM stroma, their intravasation, leading to the colonization of haematopoietic organs and the establishment of definitive haematopoiesis.

Histopathological and cognitive defects induced by Nef in the brain

Complex mechanisms of human immunodeficiency virus type‐1 (HIV‐1) brain pathogenesis suggest the contribution of individual HIV‐1 gene products. Among them, the Nef protein has been reported to harbor a major determinant of pathogenicity in AIDS‐like disease. The goal of the present study was to determine whether Nef protein expressed in vivo by primary macrophages could induce a brain toxicity also affecting the behavior of the rat. To achieve this goal we grafted Nef‐transduced macrophages into the rat hippocampus. Two months post‐transplantation, we observed that Nef induces monocyte/macrophage recruitment, expression of TNF‐α, and astrogliosis. No apoptotic event was detected. We further demonstrated that Nef neurotoxicity is associated with cognitive deficits.—Mordelet, E., Kissa, K., Cressant, A., Gray, F., Ozden, S., Vidal, C., Charneau, P., Granon, S. Histopathological and cognitive defects induced by Nef in the brain. FASEB J. 18, 1851‐1861 (2004)