A Novel Role for MAPKAPK2 in Morphogenesis during Zebrafish Development

One of the earliest morphogenetic processes in the development of many animals is epiboly. In the zebrafish, epiboly ensues when the animally localized blastoderm cells spread, thin over, and enclose the vegetally localized yolk. Only a few factors are known to function in this fundamental process. We identified a maternal-effect mutant, betty boop (bbp), which displays a novel defect in epiboly, wherein the blastoderm margin constricts dramatically, precisely when half of the yolk cell is covered by the blastoderm, causing the yolk cell to burst. Whole-blastoderm transplants and mRNA microinjection rescue demonstrate that Bbp functions in the yolk cell to regulate epiboly. We positionally cloned the maternal-effect bbp mutant gene and identified it as the zebrafish homolog of the serine-threonine kinase Mitogen Activated Protein Kinase Activated Protein Kinase 2, or MAPKAPK2, which was not previously known to function in embryonic development. We show that the regulation of MAPKAPK2 is conserved and p38 MAP kinase functions upstream of MAPKAPK2 in regulating epiboly in the zebrafish embryo. Dramatic alterations in calcium dynamics, together with the massive marginal constrictive force observed in bbp mutants, indicate precocious constriction of an F-actin network within the yolk cell, which first forms at 50% epiboly and regulates epiboly progression. We show that MAPKAPK2 activity and its regulator p38 MAPK function in the yolk cell to regulate the process of epiboly, identifying a new pathway regulating this cell movement process. We postulate that a p38 MAPKAPK2 kinase cascade modulates the activity of F-actin at the yolk cell margin circumference allowing the gradual closure of the blastopore as epiboly progresses

Consensus guidelines for the use and interpretation of angiogenesis assays

The formation of new blood vessels, or angiogenesis, is a complex process that plays important roles in growth and development, tissue and organ regeneration, as well as numerous pathological conditions. Angiogenesis undergoes multiple discrete steps that can be individually evaluated and quantified by a large number of bioassays. These independent assessments hold advantages but also have limitations. This article describes in vivo, ex vivo, and in vitro bioassays that are available for the evaluation of angiogenesis and highlights critical aspects that are relevant for their execution and proper interpretation. As such, this collaborative work is the first edition of consensus guidelines on angiogenesis bioassays to serve for current and future reference

Enhancing precision in fs-laser material processing by simultaneous spatial and temporal focusing

In recent years, femtosecond (fs)-lasers have evolved into a versatile tool for high precision micromachining of transparent materials because nonlinear absorption in the focus can result in refractive index modifications or material disruptions. However, when high pulse energies or low numerical apertures are required, nonlinear side effects such as self-focusing, filamentation or white light generation can decrease the modification quality. In this paper, we apply simultaneous spatial and temporal focusing (SSTF) to overcome these limitations. The main advantage of SSTF is that the ultrashort pulse is only formed at the focal plane, thereby confining the intensity distribution strongly to the focal volume and suppressing detrimental nonlinear side effects. Thus, we investigate the optical breakdown within a water cell by pump-probe shadowgraphy, comparing conventional focusing and SSTF under equivalent focusing conditions. The plasma formation is well confined for low pulse energies <2 µJ, but higher pulse energies lead to the filamentation and break-up of the disruptions for conventional focusing, thereby decreasing the modification quality. In contrast, plasma induced by SSTF stays well confined to the focal plane, even for high pulse energies up to 8 µJ, preventing extended filaments, side branches or break-up of the disruptions. Furthermore, while conventional focusing leads to broadband supercontinuum generation, only marginal spectral broadening is observed using SSTF. These experimental findings are in excellent agreement with numerical simulations of the nonlinear pulse propagation and interaction processes. Therefore, SSTF appears to be a powerful tool to control the processing of transparent materials, e.g., for precise ophthalmic fs-surgery

Endothelial cells dynamically compete for the tip cell position during angiogenic sprouting

Sprouting angiogenesis requires the coordinated behaviour of endothelial cells, regulated by Notch and vascular endothelial growth factor receptor (VEGFR) signalling. Here, we use computational modelling and genetic mosaic sprouting assays in vitro and in vivo to investigate the regulation and dynamics of endothelial cells during tip cell selection. We find that endothelial cells compete for the tip cell position through relative levels of Vegfr1 and Vegfr2, demonstrating a biological role for differential Vegfr regulation in individual endothelial cells. Differential Vegfr levels affect tip selection only in the presence of a functional Notch system by modulating the expression of the ligand Dll4. Time-lapse microscopy imaging of mosaic sprouts identifies dynamic position shuffling of tip and stalk cells in vitro and in vivo, indicating that the VEGFR-Dll4-Notch signalling circuit is constantly re-evaluated as cells meet new neighbours. The regular exchange of the leading tip cell raises novel implications for the concept of guided angiogenic sprouting.