Modelling human wiskott aldrich syndrome protein mutants in zebrafish larvae using live in vivo imaging

Abstract

SummaryWiskott–Aldrich syndrome (WAS) and X-linked neutropenia (XLN) are immunodeficiencies in which the function of several haematopoietic cell lineages is perturbed as a result of mutations in the actin regulator WASp. From in vitro cell biology experiments, and biochemical and structural approaches, we know much about the functional domains of WASp and how WASp might regulate the dynamic actin cytoskeleton downstream of activators such as Cdc42, but in vivo experiments are much more challenging. In patients, there is a correlation between clinical disease and genotype, with severe reductions in WASp expression or function associating with complex multilineage immunodeficiency, whereas specific mutations that cause constitutive activation of WASp result in congenital neutropenia. Here, we take advantage of the genetic tractability and translucency of zebrafish larvae to first characterise how a null mutant in zfWASp influences the behaviour of neutrophils and macrophages in response to tissue damage and to clearance of infections. We then use this mutant background to study how leukocyte lineage-specific transgenic replacement with human WASp variants (including normal wild type and point mutations that either fail to bind Cdc42 or cannot be phosphorylated, and a constitutively active mutant equivalent to that seen in XLN patients) alter the capacity for generation of neutrophils, their chemotactic response to wounds and the phagocytic clearance capacity of macrophages. This model provides a unique insight into WASp-related immunodeficiency at both a cellular and whole organism level.

Neutrophils in host defense: new insights from zebrafish

“…As in zWASp1 morphants, the zWASp1 null mutant has a normal number of neutrophils and macrophages, but these leukocytes exhibit impaired recruitment to a tailfin wound [85] with defects in pseudopod selection similar to in vitro studies [86,87]. The WASp null mutant also has increased susceptibility to bacterial infection with Staphylococcus aureus, recapitulating the infection phenotype of WAS patients [85]. Constitutively activating mutations in the GTPase binding domain of WASp result in X-linked neutropenia [88] and have also been modeled in zebrafish larvae [85] to provide an in vivo model for human X-linked neutropenia.…”

“…To validate the morpholino studies, a zWASp1 mutant was generated [83] by TILLING [84]. As in zWASp1 morphants, the zWASp1 null mutant has a normal number of neutrophils and macrophages, but these leukocytes exhibit impaired recruitment to a tailfin wound [85] with defects in pseudopod selection similar to in vitro studies [86,87]. The WASp null mutant also has increased susceptibility to bacterial infection with Staphylococcus aureus, recapitulating the infection phenotype of WAS patients [85].…”

“…The WASp null mutant also has increased susceptibility to bacterial infection with Staphylococcus aureus, recapitulating the infection phenotype of WAS patients [85]. Constitutively activating mutations in the GTPase binding domain of WASp result in X-linked neutropenia [88] and have also been modeled in zebrafish larvae [85] to provide an in vivo model for human X-linked neutropenia.…”

MicroRNAs in neutrophils: potential next generation therapeutics for inflammatory ailments

“…It is also noted that when stimulated with a strong local inflammation at a remote site, such as the otic vesicle, these neutrophils will first mobilize into the vasculature and then extravasate at the infection site where they engulf bacteria, which fully models the process in humans . Several disorders such as Leukocyte Adhesion Deficiency IV, Warts, Hypogammaglobulinemia, Immunodeficiency, Myelokathexis (WHIM) syndrome, and the Wiskott–Aldrich Syndrome are modeled in zebrafish. Not only do these models faithfully recapitulate human conditions, unique insights into those immunodeficiency disorders, particularly related to neutrophils, are provided at both the cellular and whole organism levels.…”

WASP and SCAR are evolutionarily conserved in actin-filled pseudopod-based motility

“…N-WASP is ubiquitously expressed in mammals, and is dispensable for lamellipodia or filopodia formation by adherent fibroblasts (Snapper et al, 2001;Lommel et al, 2001;Sarmiento et al, 2008), which has lead many researchers to discount a role for any WASP protein in protrusions or motility (Small and Rottner, 2010;Veltman and Insall, 2010b). Mammalian WASP, on the other hand, is expressed only in blood cells, where it has been shown to be involved in migration and pseudopod formation (Jones et al, 2002;Burns et al, 2001;Badolato et al, 1998;Jones et al, 2013;Shi et al, 2009;Ishihara et al, 2012). Further evidence for a role of WASP in cell migration comes from the handful of papers studying WASP in non-mammalian cells (Veltman et al, 2012;Zhu et al, 2016).…”