Adenosine signaling promotes hematopoietic stem and progenitor cell emergence.

Hematopoietic stem cells (HSCs) emerge from aortic endothelium via the endothelial-to-hematopoietic transition (EHT). The molecular mechanisms that initiate and regulate EHT remain poorly understood. Here, we show that adenosine signaling regulates hematopoietic stem and progenitor cell (HSPC) development in zebrafish embryos. The adenosine receptor A2b is expressed in the vascular endothelium before HSPC emergence. Elevated adenosine levels increased runx1(+)/cmyb(+) HSPCs in the dorsal aorta, whereas blocking the adenosine pathway decreased HSPCs. Knockdown of A2b adenosine receptor disrupted scl(+) hemogenic vascular endothelium and the subsequent EHT process. A2b adenosine receptor activation induced CXCL8 via cAMP-protein kinase A (PKA) and mediated hematopoiesis. We further show that adenosine increased multipotent progenitors in a mouse embryonic stem cell colony-forming assay and in embryonic day 10.5 aorta-gonad-mesonephros explants. Our results demonstrate that adenosine signaling plays an evolutionary conserved role in the first steps of HSPC formation in vertebrates.

In vivo imaging and characterization of actin microridges.

Actin microridges form labyrinth like patterns on superficial epithelial cells across animal species. This highly organized assembly has been implicated in mucus retention and in the mechanical structure of mucosal surfaces, however the mechanisms that regulate actin microridges remain largely unknown. Here we characterize the composition and dynamics of actin microridges on the surface of zebrafish larvae using live imaging. Microridges contain phospho-tyrosine, cortactin and VASP, but not focal adhesion kinase. Time-lapse imaging reveals dynamic changes in the length and branching of microridges in intact animals. Transient perturbation of the microridge pattern occurs before cell division with rapid re-assembly during and after cytokinesis. Microridge assembly is maintained with constitutive activation of Rho or inhibition of myosin II activity. However, expression of dominant negative RhoA or Rac alters microridge organization, with an increase in distance between microridges. Latrunculin A treatment and photoconversion experiments suggest that the F-actin filaments are actively treadmilling in microridges. Accordingly, inhibition of Arp2/3 or PI3K signaling impairs microridge structure and length. Taken together, actin microridges in zebrafish represent a tractable in vivo model to probe pattern formation and dissect Arp2/3-mediated actin dynamics in vivo.

Localized bacterial infection induces systemic activation of neutrophils through Cxcr2 signaling in zebrafish.

Neutrophils are the first line of defense against tissue damage and are rapidly mobilized to sites of bacterial infection. However, the signals that regulate neutrophil recruitment are not well defined. Here, using photolabel-enabled fate mapping in zebrafish larvae, we show that localized otic infection with Pseudomonas aeruginosa induces systemic activation and mobilization of neutrophils from the CHT through Cxcr2 signaling. We have cloned the zebrafish Cxcr1 and Cxcr2 receptors and show that Cxcr2 functions as a Cxcl8 receptor in live zebrafish. With the use of morpholino-mediated depletion, we show that infection-induced neutrophil mobilization from the CHT is mediated by Cxcr2 but not Cxcr1. By contrast, Cxcr2 depletion does not affect neutrophil recruitment to the chemoattractant LTB4. Taken together, our findings identify Cxcl8-Cxcr2 signaling as an infection-induced long-range cue that mediates neutrophil motility and mobilization from hematopoietic tissues, positioning Cxcr2 as a critical pathway that mediates infection-induced systemic activation of neutrophils.

Zebrafish Entrapment By Restriction Array (ZEBRA) device: a low-cost, agarose-free zebrafish mounting technique for automated imaging.

The zebrafish has emerged as a useful model system for a variety of studies, including the investigation of inflammation and immunity. However, current zebrafish imaging techniques, such as agraose mounting, can be time-consuming and detrimental for long-term imaging. Alternatively, automated sorting and imaging systems can be costly and/or complicated to assemble. Here we describe the Zebrafish Entrapment by Restriction Array (ZEBRA) device, a microfluidic device that can be used to quickly and repeatably position zebrafish embryos in a predictable array using only a pipette. This technique is well suited for use with automated microscope stages leading to decreased imaging time and increased throughput compared to traditional methods. The addition of access ports above the embryo can be used to administer treatments, and potentially wounding or injections. We demonstrate the effectiveness of this device for a neutrophil migration screening application using larvae 3 days post fertilization (dpf) Tg(mpx:dendra2). Larvae were loaded into ZEBRA devices and treated with a neutrophil attractant (LTB4) or LTB4 with and without a PI3K inhibitor, LY294002. Treatment with LY294002 impaired neutrophil motility into the fin induced by LTB4 treatment. The findings report the development of ZEBRA a device that can be used to screen for small molecules that affect leukocyte motility and inflammation using live zebrafish.

Innate immune response to Streptococcus iniae infection in zebrafish larvae.

Streptococcus iniae causes systemic infection characterized by meningitis and sepsis. Here, we report a larval zebrafish model of S. iniae infection. Injection of wild-type S. iniae into the otic vesicle induced a lethal infection by 24 h postinfection. In contrast, an S. iniae mutant deficient in polysaccharide capsule (cpsA mutant) was not lethal, with greater than 90% survival at 24 h postinfection. Live imaging demonstrated that both neutrophils and macrophages were recruited to localized otic infection with mutant and wild-type S. iniae and were able to phagocytose bacteria. Depletion of neutrophils and macrophages impaired host survival following infection with wild-type S. iniae and the cpsA mutant, suggesting that leukocytes are critical for host survival in the presence of both the wild-type and mutant bacteria. However, zebrafish larvae with impaired neutrophil function but normal macrophage function had increased susceptibility to wild-type bacteria but not the cpsA mutant. Taking these findings together, we have developed a larval zebrafish model of S. iniae infection and have found that although neutrophils are important for controlling infection with wild-type S. iniae, neutrophils are not necessary for host defense against the cpsA mutant.

The SH2-domain-containing inositol 5-phosphatase (SHIP) limits the motility of neutrophils and their recruitment to wounds in zebrafish.

Neutrophil recruitment to sites of injury or infection is essential for host defense, but it needs to be tightly regulated to prevent tissue damage. Phosphoinositide 3-kinase (PI3K), which generates the phosphatidylinositol (3,4,5)-trisphosphate [PI(3,4,5)P(3)], is necessary for neutrophil motility in vivo; however, the role of SH2-domain-containing 5-inositol phosphatase (SHIP) enzymes, which hydrolyze PI(3,4,5)P(3) to phosphatidylinositol 3,4-bisphosphate [PI(3,4)P(2)], is not well understood. Here we show that SHIP phosphatases limit neutrophil motility in live zebrafish. Using real-time imaging of bioprobes specific for PI(3,4,5)P(3) and PI(3,4)P(2) in neutrophils, we found that PI(3,4,5)P(3) and PI(3,4)P(2) accumulate at the leading edge while PI(3,4)P(2) also localizes to the trailing edge of migrating neutrophils in vivo. Depletion of SHIP phosphatases using morpholino oligonucleotides led to increased neutrophil 3D motility and neutrophil infiltration into wounds. The increase in neutrophil wound recruitment in SHIP morphants was rescued by treatment with low dose PI3Kγ inhibitor, suggesting that SHIP limits neutrophil motility by modulating PI3K signaling. Moreover, overexpression of the SHIP phosphatase domain in neutrophils impaired neutrophil 3D migration. Taken together, our findings suggest that SHIP phosphatases control neutrophil inflammation by limiting neutrophil motility in vivo.

Dual roles for Rac2 in neutrophil motility and active retention in zebrafish hematopoietic tissue.

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. Noninvasive 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 warts, hypogammaglobulinemia, infections, and myelokathexis (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.

Live imaging of neutrophil motility in a zebrafish model of WHIM syndrome.

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, hypo-gammaglobulinemia, 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 CXCR4-SDF1 interactions in regulating neutrophil motility in vivo is not known. Here, we use the optical transparency of zebrafish to visualize neutrophil trafficking in vivo in a zebrafish model of WHIM syndrome. We demonstrate that expression of WHIM mutations in zebrafish neutrophils induces neutrophil retention in hematopoietic tissue, impairing neutrophil motility and wound recruitment. The neutrophil retention signal induced by WHIM truncation mutations is SDF1 dependent, because depletion of SDF1 with the use of morpholino oligonucleotides restores neutrophil chemotaxis to wounds. Moreover, localized activation of a genetically encoded, photoactivatable Rac guanosine triphosphatase is sufficient to direct migration of neutrophils that express the WHIM mutation. The findings suggest that this transgenic zebrafish model of WHIM syndrome may provide a valuable tool to screen for agents that modify CXCR4-SDF1 retention signals.

Development and evaluation of methods for structured recording of heart murmur findings using SNOMED-CT post-coordination.

OBJECTIVE: This study evaluated an existing SNOMED-CT model for structured recording of heart murmur findings and compared it to a concept-dependent attributes model using content from SNOMED-CT. METHODS: The authors developed a model for recording heart murmur findings as an alternative to SNOMED-CT's use of Interprets and Has interpretation. A micro-nomenclature was then created to support each model using subset and extension mechanisms described for SNOMED-CT. Each micro-nomenclature included a partonomy of cardiac cycle timing values. A mechanism for handling ranges of values was also devised. One hundred clinical heart murmurs were recorded using purpose-built recording software based on both models. RESULTS: Each micro-nomenclature was extended through the addition of the same list of concepts. SNOMED role grouping was required in both models. All 100 clinical murmurs were described using each model. The only major differences between the two models were the number of relationship rows required for storage and the hierarchical assignments of concepts within the micro-nomenclatures. CONCLUSION: The authors were able to capture 100 clinical heart murmurs with both models. Requirements for implementing the two models were virtually identical. In fact, data stored using these models could be easily interconverted. There is no apparent penalty for implementing either approach.