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In vitro, depending on extracellular matrix (ECM) architecture, macrophages migrate either in amoeboid or mesenchymal mode; while the first is a general trait of leukocytes, the latter is associated with tissue remodelling via Matrix Metalloproteinases (MMPs). To assess whether these stereotyped migrations could be also observed in a physiological context, we used the zebrafish embryo and monitored macrophage morphology, behaviour and capacity to mobilise haematopoietic stem/progenitor cells (HSPCs), as a final functional readout. Morphometric analysis identified 4 different cell shapes. Live imaging revealed that macrophages successively adopt all four shapes as they migrate through ECM. Treatment with inhibitors of MMPs or Rac GTPase to abolish mesenchymal migration, suppresses both ECM degradation and HSPC mobilisation while differently affecting macrophage behaviour. This study depicts real time macrophage behaviour in a physiological context and reveals extreme reactivity of these cells constantly adapting and switching migratory shapes to achieve HSPCs proper mobilisation. [ABSTRACT FROM AUTHOR]

All blood cells originate from hematopoietic stem/progenitor cells (HSPCs). HSPCs are formed from endothelial cells (ECs) of the dorsal aorta (DA), via endothelial-to-hematopoietic transition (EHT). The zebrafish is a primary model organism to study the process in vivo. While the role of mechanical stress in controlling gene expression promoting cell differentiation is actively investigated, mechanisms driving shape changes of the DA and individual ECs remain poorly understood. We address this problem by developing a new DA micromechanical model and applying it to experimental data on zebrafish morphogenesis. The model considers the DA as an isotropic tubular membrane subjected to hydrostatic blood pressure and axial stress. The DA evolution is described as a movement in the dimensionless controlling parameters space: normalized hydrostatic pressure and axial stress. We argue that HSPC production is accompanied by two mechanical instabilities arising in the system due to the plane stress in the DA walls and show how a complex interplay between mechanical forces in the system drives the emerging morphological changes. [ABSTRACT FROM AUTHOR]

During embryogenesis of all vertebrates, haematopoietic stem/progenitor cells (HSPCs) extrude from the aorta by a complex process named endothelial-to-haematopoietic transition (EHT). HSPCs will then colonize haematopoietic organs allowing haematopoiesis throughout adult life. The mechanism underlying EHT including the role of each aortic endothelial cell (EC) within the global aorta dynamics remains unknown. In the present study, we show for the first time that EHT involves the remodelling of individual cells within a collective migration of ECs which is tightly orchestrated, resulting in HSPCs extrusion in the sub-aortic space without compromising aorta integrity. By performing a cross-disciplinary study which combines high-resolution 4D imaging and theoretical analysis based on the concepts of classical mechanics, we propose that this complex developmental process is dependent on mechanical instabilities of the aorta preparing and facilitating the extrusion of HSPCs. [ABSTRACT FROM AUTHOR]

Identifying genes involved in vertebrate developmental processes and characterizing this involvement are daunting tasks, especially in the mouse where viviparity complicates investigations. Attempting to devise a streamlined approach for this type of study we focused on limb development. We cultured E10.5 and E12.5 embryos and performed transcriptional profiling to track molecular changes in the forelimb bud over a 6-hour time-window. The expression of certain genes was found to diverge rapidly from its normal path, possibly reflecting the activation of a stress-induced response. Others, however, maintained for up to 3 hours dynamic expression profiles similar to those seen in utero. Some of these resilient genes were known regulators of limb development. The implication of the others in this process was either unsuspected or unsubstantiated. The localized knockdown of two such genes, Fgf11 and Tbx1, hampered forelimb bud development, providing evidence of their implication. These results show that combining embryo culture, transcriptome analysis and RNA interference could speed up the identification of genes involved in a variety of developmental processes, and the validation of their implication. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Parabiosis, the surgical generation of conjoined organisms sharing a common bloodstream, has been a powerful tool for studying hematopoietic cell migration and interaction with stromal niches in rodent and avian systems. We describe a technique to generate parabiotic zebrafish embryos based on blastula fusion. This procedure permits the in vivo visualization of hematopoietic cell migration and homing to niches and peripheral tissues in zebrafish parabiotes of different genetic backgrounds. [ABSTRACT FROM AUTHOR]