SummaryTissue wounding induces the rapid recruitment of leukocytes1. Wounds and tumors, a type of “unhealed wound”2, generate hydrogen peroxide (H2O2) through a NADPH oxidase (NOX) and the extracellular H2O2 mediates recruitment of leukocytes, particularly first responders of innate immunity, neutrophils, to injured tissue3–6. However, it is not known what sensor neutrophils use to detect the redox state at wounds. Here we identify the Src family kinase (SFK) Lyn as a redox sensor that mediates initial neutrophil recruitment to wounds in zebrafish larvae. Lyn activation in neutrophils is dependent on wound-derived H2O2 following tissue injury and inhibition of Lyn attenuates neutrophil wound recruitment. Inhibition of SFKs also disrupted H2O2-mediated chemotaxis of primary human neutrophils. In vitro analysis identified a single cysteine residue, C466, as being responsible for direct oxidation-mediated activation of Lyn. Furthermore, transgenic tissue-specific reconstitution with wild-type Lyn and a cysteine mutant revealed that Lyn C466 is important for the neutrophil wound response and downstream signaling in vivo. This is the first identification, to our knowledge, of a physiological redox sensor that mediates leukocyte wound attraction in multicellular organisms.

Summary Cell polarity is crucial for directed migration. Here we show that phosphoinositide 3-kinase (PI(3)K) mediates neutrophil migration in vivo by differentially regulating cell protrusion and polarity. The dynamics of PI(3)K products PI(3,4,5)P3-PI(3,4)P2 during neutrophil migration were visualized in living zebrafish, revealing that PI(3)K activation at the leading edge is critical for neutrophil motility in intact tissues. A genetically encoded photoactivatable Rac was used to demonstrate that localized activation of Rac is sufficient to direct migration with precise temporal and spatial control in vivo. Similar stimulation of PI(3)K-inhibited cells did not direct migration. Localized Rac activation rescued membrane protrusion but not anteroposterior polarization of F-actin dynamics of PI(3)K-inhibited cells. Uncoupling Rac-mediated protrusion and polarization suggests a paradigm of two-tiered PI(3)K-mediated regulation of cell motility. This work provides new insight into how cell signaling at the front and back of the cell is coordinated during polarized cell migration in intact tissues within a multicellular organism.

The receptor tyrosine kinase Ror2 plays important roles in developmental morphogenesis. It has recently been shown that Ror2 mediates Wnt5a-induced noncanonical Wnt signaling by activating the Wnt–JNK pathway and inhibiting the β-catenin–TCF pathway. However, the function of Ror2 in noncanonical Wnt signaling leading to cell migration is largely unknown. We show, using genetically different or manipulated cultured cells, that Ror2 is critical for Wnt5a-induced, but not Wnt3a-induced, cell migration. Ror2-mediated cell migration requires the extracellular cysteine-rich domain (CRD), which is the binding site for Wnt5a, and the cytoplasmic proline-rich domain (PRD) of Ror2. Furthermore, Ror2 can mediate filopodia formation via actin reorganization, irrespective of Wnt5a, and this Ror2-mediated filopodia formation requires the actin-binding protein filamin A, which associates with the PRD of Ror2. Intriguingly, disruption of filopodia formation by suppressing the expression of either Ror2 or filamin A inhibits Wnt5a-induced cell migration, indicating that Ror2-mediated filopodia formation is essential for Wnt5a-induced cell migration.

Redox, SFK, and calcium signaling are immediate “wound signals” that integrate early wound responses and late epimorphic regeneration.

Summary Neutrophil homeostasis is essential for host defense. Here we identify dual roles for Rac2 during neutrophil homeostasis using a zebrafish model of primary immune deficiency induced by the human inhibitory Rac2D57N mutation in neutrophils. Non-invasive live imaging of Rac2 morphants or Rac2D57N zebrafish larvae demonstrates an essential role for Rac2 in regulating 3D motility and the polarization of F-actin dynamics and PI(3)K signaling in vivo. Tracking of photolabeled Rac2-deficient neutrophils from hematopoietic tissue also shows increased mobilization into the circulation, indicating that neutrophil mobilization does not require traditionally defined cell motility. Moreover, excessive neutrophil retention in hematopoietic tissue resulting from a constitutively-active CXCR4 mutation in zebrafish WHIM syndrome is partially rescued by the inhibitory Rac2 mutation. These findings reveal that Rac2 signaling is necessary for both neutrophil 3D motility and CXCR4-mediated neutrophil retention in hematopoietic tissue, thereby limiting neutrophil mobilization, a critical first step in the innate immune response.

How neutrophils traffic during inflammation in vivo remains elusive. To visualize the origin and fate of neutrophils during induction and resolution of inflammation, we established a genetically encoded photolabeling system by generating transgenic zebrafish that express a photoconvertible fluorescent reporter Dendra2 in neutrophils. Spatiotemporal photolabeling of neutrophils in vivo demonstrates that they emerge from the hematopoietic tissue in close proximity to injured tissue and repeat forward and reverse migration between the wound and the vasculature. Subsequently, neutrophils disperse throughout the body as wound-healing proceeds, contributing to local resolution at injured tissue and systemic dissemination of wound-sensitized neutrophils. Tissue damage also alters the fate of neutrophils in the caudal hematopoietic tissue and promotes caudorostral mobilization of neutrophils via the circulation to the cephalic mesenchyme. This work provides new insight into neutrophil behaviors during inflammation and resolution within a multicellular organism.

CXCR4 is a G protein-coupled chemokine receptor that has been implicated in the pathogenesis of primary immunodeficiency disorders and cancer. Autosomal dominant gain-of-function truncations of CXCR4 are associated with warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome, a primary immunodeficiency disorder characterized by neutropenia and recurrent infections. Recent progress has implicated CXCR4-SDF1 (stromal cell-derived factor 1) signaling in regulating neutrophil homeostasis, but the precise role of IntroductionStromal cell-derived factor 1 (SDF1, CXCL12)-mediated activation 1 of the chemokine receptor CXCR4 is important for both normal and pathologic processes, including primordial germ cell migration, HIV pathogenesis, invasive migration of cancer cells, and leukocyte trafficking. 2-5 Therefore, there is substantial interest in understanding how CXCR4-SDF1 signaling regulates cell motility and how these mechanisms can be targeted to treat human disease. CXCR4 signaling is attenuated by receptor internalization, which is regulated by phosphorylation events and binding of regulatory proteins to the cytoplasmic tail. 6 The functional importance of CXCR4 internalization is highlighted by the dominantly inherited primary immunodeficiency, warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome, in which truncations of CXCR4 lead to altered signaling and gain of function. [7][8][9] WHIM syndrome is characterized by warts, hypogammaglobulinemia, infections, and myelokathexis, a severe chronic neutropenia. 10,11 Substantial evidence supports the importance of CXCR4 signaling in regulating neutrophil homeostasis and release from the bone marrow (BM). 5 It has been postulated that the neutropenia in patients with WHIM syndrome results from both neutrophil retention in the BM and enhanced neutrophil apoptosis of retained neutrophils. 11 Direct evidence to support this hypothesis has been provided by a mouse model of WHIM syndrome induced by the ectopic expression of WHIM truncation mutations of CXCR4 in hematopoietic stem cells that show impaired neutrophil release into the blood and increased rates of apoptosis in the BM. 12 Previous reports indicate that neutrophils from patients with WHIM show increased signaling 13 and chemotaxis 8,9 in response to SDF1. However, some reports have suggested that the C-terminus of CXCR4 can both positively and negatively regulate cell motility 8,9,14 and, alternatively, may be involved in modulating the precise targeting of cells in vivo. 15 Despite the importance of CXCR4-SDF1 signaling, few animal models of WHIM syndrome are amenable to imaging or screening for drugs that modulate CXCR4-SDF1 function in vivo. Modeling WHIM syndrome is particularly attractive because CXCR4 signaling is important to many disease processes and is a direct result of aberrant chemokine signaling. Therefore, developing a model of WHIM syndrome in a system that allows the direct visualization of motility and chemotactic events in vivo would be a beneficial ...

Zebrafish have emerged as a powerful model system to study leukocyte recruitment and inflammation. Here we characterize the morphology and function of inflammatory macrophages in zebrafish larvae. These macrophages can be distinguished from neutrophils by immunolabeling of L-Plastin without MPO co-expression and by an elongated morphology. Live imaging of transgenic zMPO:GFP larvae demonstrate that GFPlo macrophages migrate to wounds by extension of thin pseudopods and carry out phagocytosis of tissue debris, and FACS analysis of leukocyte markers indicates expression of CSF1R in these macrophages. These findings identify distinct functional and morphological characteristics of inflammatory macrophages in zebrafish larvae.