Cell segregation and border sharpening by Eph receptor-ephrin-mediated heterotypic repulsion.

Eph receptor and ephrin signalling has a major role in cell segregation and border formation, and may act through regulation of cell adhesion, repulsion or tension. To elucidate roles of cell repulsion and adhesion, we combined experiments in cell culture assays with quantitations of cell behaviour which are used in computer simulations. Cells expressing EphB2, or kinase-inactive EphB2 (kiEphB2), segregate and form a sharp border with ephrinB1-expressing cells, and this is disrupted by knockdown of N-cadherin. Measurements of contact inhibition of locomotion reveal that EphB2-, kiEphB2- and ephrinB1-expressing cells have strong heterotypic and weak homotypic repulsion. EphB2 cells have a transient increase in migration after heterotypic activation, which underlies a shift in the EphB2-ephrinB1 border but is not required for segregation or border sharpening. Simulations with the measured values of cell behaviour reveal that heterotypic repulsion can account for cell segregation and border sharpening, and is more efficient than decreased heterotypic adhesion. By suppressing homotypic repulsion, N-cadherin creates a sufficient difference between heterotypic and homotypic repulsion, and enables homotypic cohesion, both of which are required to sharpen borders.

Inference of random walk models to describe leukocyte migration.

While the majority of cells in an organism are static and remain relatively immobile in their tissue, migrating cells occur commonly during developmental processes and are crucial for a functioning immune response. The mode of migration has been described in terms of various types of random walks. To understand the details of the migratory behaviour we rely on mathematical models and their calibration to experimental data. Here we propose an approximate Bayesian inference scheme to calibrate a class of random walk models characterized by a specific, parametric particle re-orientation mechanism to observed trajectory data. We elaborate the concept of transition matrices (TMs) to detect random walk patterns and determine a statistic to quantify these TM to make them applicable for inference schemes. We apply the developed pipeline to in vivo trajectory data of macrophages and neutrophils, extracted from zebrafish that had undergone tail transection. We find that macrophage and neutrophils exhibit very distinct biased persistent random walk patterns, where the strengths of the persistence and bias are spatio-temporally regulated. Furthermore, the movement of macrophages is far less persistent than that of neutrophils in response to wounding.

P38 and JNK have opposing effects on persistence of in vivo leukocyte migration in zebrafish.

The recruitment and migration of macrophages and neutrophils is an important process during the early stages of the innate immune system in response to acute injury. Transgenic pu.1:EGFP zebrafish permit the acquisition of leukocyte migration trajectories during inflammation. Currently, these high-quality live-imaging data are mainly analysed using general statistics, for example, cell velocity. Here, we present a spatio-temporal analysis of the cell dynamics using transition matrices, which provide information of the type of cell migration. We find evidence that leukocytes exhibit types of migratory behaviour, which differ from previously described random walk processes. Dimethyl sulfoxide treatment decreased the level of persistence at early time points after wounding and ablated temporal dependencies observed in untreated embryos. We then use pharmacological inhibition of p38 and c-Jun N-terminal kinase mitogen-activated protein kinases to determine their effects on in vivo leukocyte migration patterns and discuss how they modify the characteristics of the cell migration process. In particular, we find that their respective inhibition leads to decreased and increased levels of persistent motion in leukocytes following wounding. This example shows the high level of information content, which can be gained from live-imaging data if appropriate statistical tools are used.

Incorporation of an experimentally determined MTF for spatial frequency filtering and deconvolution during optical projection tomography reconstruction.

We demonstrate two techniques to improve the quality of reconstructed optical projection tomography (OPT) images using the modulation transfer function (MTF) as a function of defocus experimentally determined from tilted knife-edge measurements. The first employs a 2-D binary filter based on the MTF frequency cut-off as an additional filter during back-projection reconstruction that restricts the high frequency information to the region around the focal plane and progressively decreases the spatial frequency bandwidth with defocus. This helps to suppress "streak" artifacts in OPT data acquired at reduced angular sampling, thereby facilitating faster OPT acquisitions. This method is shown to reduce the average background by approximately 72% for an NA of 0.09 and by approximately 38% for an NA of 0.07 compared to standard filtered back-projection. As a biological illustration, a Fli:GFP transgenic zebrafish embryo (3 days post-fertilisation) was imaged to demonstrate the improved imaging speed (a quarter of the acquisition time). The second method uses the MTF to produce an appropriate deconvolution filter that can be used to correct for the spatial frequency modulation applied by the imaging system.

The NOTCH pathway contributes to cell fate decision in myelopoiesis.

BACKGROUND: Controversy persists regarding the role of Notch signaling in myelopoiesis. We have used genetic approaches, employing two Notch zebrafish mutants deadly seven (DES) and beamter (BEA) with disrupted function of notch1a and deltaC, respectively, and Notch1a morphants to analyze the development of leukocyte populations in embryonic and mature fish. DESIGN AND METHODS: Myelomonocytes were quantified in early embryos by in situ hybridization using a myeloper-oxidase (mpx) probe. Morpholinos were used to knock down expression of Notch1a or DeltaC. Wound healing assays and/or flow cytometry were used to quantify myelomonocytes in 5-day post-fertilization (dpf) Notch mutants (BEA and DES), morphants or pu.1:GFP, mpx:GFP and fms:RFP transgenic embryos. Flow cytometry was performed on 2-3 month old mutant fish. RESULTS: The number of mpx(+) cells in embryos was reduced at 48 hpf (but not at 26 hpf) in DES compared to WT. At 5 dpf this was reflected by a reduction in the number of myelomonocytic cells found at the wound site in mutants and in Notch1a morphants. This was due to a reduced number of myelomonocytes developing rather than a deficit in the migratory ability since transient inhibition of Notch signaling using DAPT had no effect. The early deficit in myelopoiesis was maintained into later life, 2-3 month old BEA and DES fish having a decreased proportion of myelomonocytes in both the hematopoietic organ (kidney marrow) and the periphery (coelomic cavity). CONCLUSIONS: Our results indicate that defects in Notch signaling affect definitive hematopoiesis, altering myelopoiesis from the early stages of development into the adult.

In vivo fluorescence lifetime optical projection tomography.

We demonstrate the application of fluorescence lifetime optical projection tomography (FLIM-OPT) to in vivo imaging of lysC:GFP transgenic zebrafish embryos (Danio rerio). This method has been applied to unambiguously distinguish between the fluorescent protein (GFP) signal in myeloid cells from background autofluorescence based on the fluorescence lifetime. The combination of FLIM, an inherently ratiometric method, in conjunction with OPT results in a quantitative 3-D tomographic technique that could be used as a robust method for in vivo biological and pharmaceutical research, for example as a readout of Förster resonance energy transfer based interactions.

Skin exposure to micro- and nano-particles can cause haemostasis in zebrafish larvae.

Low mass ambient exposure to airborne particles is associated with atherothrombotic events that may be a consequence of the combustion-derived nanoparticle content. There is concern also over the potential cardiovascular impact of manufactured nanoparticles. To better understand the mechanism by which toxic airborne particles can affect cardiovascular function we utilised zebrafish as a genetically tractable model. Using light and confocal fluorescence video-microscopy, we measured heart-rate and blood flow in the dorsal aorta and caudal artery of zebrafish larvae that had been exposed to a number of toxic and non-toxic microparticles and nanoparticles. Diesel exhaust particles (DEP), carboxy-charged Latex beads (carboxy-beads) and toxic alumina (Taimicron TM300), but not non-toxic alumina (Baikalox A125), were found to promote both skin and gut cell damage, increased leukocyte invasion into the epidermis, tail muscle ischaemia and haemostasis within the caudal artery of free swimming zebrafish larvae. The presence of sodium sulfite, a reducing agent, or warfarin, an anticoagulant, within the system water abrogated the effects of both toxic alumina and carboxy-beads but not DEP. Genetic manipulation of skin barrier function augmented skin damage and haemostasis, even for the non-toxic alumina. The toxic effects of carboxy-beads were still apparent after leukocyte numbers were depleted with anti-Pu.1 morpholino. We conclude that particle uptake across skin epithelium and gut mucosal barriers, or the presence of leukocytes, is not required for particle-induced haemostasis while a compromised skin barrier function accentuated tissue injury and haemostasis.