Wiskott-Aldrich syndrome protein (WASp) is haematopoietically restricted, and is the causative protein underlying a severe human disorder that can lead to death due to immunodeficiency and haemorrhaging. Much is known about the biochemistry of WASp and the migratory capacity of WASp-defective cells in vitro, but in vivo studies of immune-cell behaviour are more challenging. Using the translucency of zebrafish larvae, we live-imaged the effects of morpholino knockdown of WASp1 (also known as Was) on leukocyte migration in response to a wound. In embryos at 22 hours post-fertilisation, primitive macrophages were impaired in their migration towards laser wounds. Once a circulatory system had developed, at 3 days post-fertilisation, we observed significantly reduced recruitment of neutrophils and macrophages to ventral fin wounds. Cell-tracking studies indicated that fewer leukocytes leave the vessels adjacent to a wound and those that do exhibit impaired navigational capacity. Their cell morphology appears unaltered but their choice of leading-edge pseudopodia is more frequently incorrect, leading to impaired chemotaxis. We also identified two zebrafish mutants in WASp1 by TILLING, one of which was in the WIP-binding domain that is the hotspot for human lesions, and mutants exhibited the same deficiencies in wound inflammation and thrombus formation as WASp1 morphants.

Navigating growth cones need to integrate, process and respond to guidance signals, requiring dynamic information transfer within and between different compartments. Studies have shown that, faced with different navigation challenges, growth cones display dynamic changes in growth kinetics and morphologies. However, it remains unknown whether these are paralleled by differences in their internal molecular dynamics. To examine whether there are protein mobility differences during guidance, we developed multiphoton fluorescence recovery after photobleaching methods to determine molecular diffusion rates in pathfinding growth cones in vivo. Actively navigating growth cones (leaders) have consistently longer recovery times than growth cones that are fasciculated and less actively navigating (followers). Pharmacological perturbations of the cytoskeleton point to actin as the primary modulator of diffusion in differently behaving growth cones. This approach provides a powerful means to quantify mobility of specific proteins in neurons in vivo and reveals that diffusion is important during axon navigation.cytoplasmic dynamics ͉ neuronal migration ͉ two-photon microscopy ͉ zebrafish

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A report on the British Societies for Cell Biology and Developmental Biology Joint Spring Meeting, University of Warwick, Coventry, UK, 6-9 April 2005.