ß-glucan-dependent shuttling of conidia from neutrophils to macrophages occurs during fungal infection establishment.

The initial host response to fungal pathogen invasion is critical to infection establishment and outcome. However, the diversity of leukocyte-pathogen interactions is only recently being appreciated. We describe a new form of interleukocyte conidial exchange called "shuttling." In Talaromyces marneffei and Aspergillus fumigatus zebrafish in vivo infections, live imaging demonstrated conidia initially phagocytosed by neutrophils were transferred to macrophages. Shuttling is unidirectional, not a chance event, and involves alterations of phagocyte mobility, intercellular tethering, and phagosome transfer. Shuttling kinetics were fungal-species-specific, implicating a fungal determinant. ß-glucan serves as a fungal-derived signal sufficient for shuttling. Murine phagocytes also shuttled in vitro. The impact of shuttling for microbiological outcomes of in vivo infections is difficult to specifically assess experimentally, but for these two pathogens, shuttling augments initial conidial redistribution away from fungicidal neutrophils into the favorable macrophage intracellular niche. Shuttling is a frequent host-pathogen interaction contributing to fungal infection establishment patterns.

Macrophages protect Talaromyces marneffei conidia from myeloperoxidase-dependent neutrophil fungicidal activity during infection establishment in vivo.

Neutrophils and macrophages provide the first line of cellular defence against pathogens once physical barriers are breached, but can play very different roles for each specific pathogen. This is particularly so for fungal pathogens, which can occupy several niches in the host. We developed an infection model of talaromycosis in zebrafish embryos with the thermally-dimorphic intracellular fungal pathogen Talaromyces marneffei and used it to define different roles of neutrophils and macrophages in infection establishment. This system models opportunistic human infection prevalent in HIV-infected patients, as zebrafish embryos have intact innate immunity but, like HIV-infected talaromycosis patients, lack a functional adaptive immune system. Importantly, this new talaromycosis model permits thermal shifts not possible in mammalian models, which we show does not significantly impact on leukocyte migration, phagocytosis and function in an established Aspergillus fumigatus model. Furthermore, the optical transparency of zebrafish embryos facilitates imaging of leukocyte/pathogen interactions in vivo. Following parenteral inoculation, T. marneffei conidia were phagocytosed by both neutrophils and macrophages. Within these different leukocytes, intracellular fungal form varied, indicating that triggers in the intracellular milieu can override thermal morphological determinants. As in human talaromycosis, conidia were predominantly phagocytosed by macrophages rather than neutrophils. Macrophages provided an intracellular niche that supported yeast morphology. Despite their minor role in T. marneffei conidial phagocytosis, neutrophil numbers increased during infection from a protective CSF3-dependent granulopoietic response. By perturbing the relative abundance of neutrophils and macrophages during conidial inoculation, we demonstrate that the macrophage intracellular niche favours infection establishment by protecting conidia from a myeloperoxidase-dependent neutrophil fungicidal activity. These studies provide a new in vivo model of talaromycosis with several advantages over previous models. Our findings demonstrate that limiting T. marneffei's opportunity for macrophage parasitism and thereby enhancing this pathogen's exposure to effective neutrophil fungicidal mechanisms may represent a novel host-directed therapeutic opportunity.

The Pu.1 target gene Zbtb11 regulates neutrophil development through its integrase-like HHCC zinc finger.

In response to infection and injury, the neutrophil population rapidly expands and then quickly re-establishes the basal state when inflammation resolves. The exact pathways governing neutrophil/macrophage lineage outputs from a common granulocyte-macrophage progenitor are still not completely understood. From a forward genetic screen in zebrafish, we identify the transcriptional repressor, ZBTB11, as critical for basal and emergency granulopoiesis. ZBTB11 sits in a pathway directly downstream of master myeloid regulators including PU.1, and TP53 is one direct ZBTB11 transcriptional target. TP53 repression is dependent on ZBTB11 cys116, which is a functionally critical, metal ion-coordinating residue within a novel viral integrase-like zinc finger domain. To our knowledge, this is the first description of a function for this domain in a cellular protein. We demonstrate that the PU.1-ZBTB11-TP53 pathway is conserved from fish to mammals. Finally, Zbtb11 mutant rescue experiments point to a ZBTB11-regulated TP53 requirement in development of other organs.

The Asymmetric Cell Division Regulators Par3, Scribble and Pins/Gpsm2 Are Not Essential for Erythroid Development or Enucleation.

Erythroid enucleation is the process by which the future red blood cell disposes of its nucleus prior to entering the blood stream. This key event during red blood cell development has been likened to an asymmetric cell division (ACD), by which the enucleating erythroblast divides into two very different daughter cells of alternate molecular composition, a nucleated cell that will be removed by associated macrophages, and the reticulocyte that will mature to the definitive erythrocyte. Here we investigated gene expression of members of the Par, Scribble and Pins/Gpsm2 asymmetric cell division complexes in erythroid cells, and functionally tested their role in erythroid enucleation in vivo and ex vivo. Despite their roles in regulating ACD in other contexts, we found that these polarity regulators are not essential for erythroid enucleation, nor for erythroid development in vivo. Together our results put into question a role for cell polarity and asymmetric cell division in erythroid enucleation.

Calcium Signaling Is Required for Erythroid Enucleation.

Although erythroid enucleation, the property of erythroblasts to expel their nucleus, has been known for 7ore than a century, surprisingly little is known regarding the molecular mechanisms governing this unique developmental process. Here we show that similar to cytokinesis, nuclear extrusion requires intracellular calcium signaling and signal transduction through the calmodulin (CaM) pathway. However, in contrast to cytokinesis we found that orthochromatic erythroblasts require uptake of extracellular calcium to enucleate. Together these functional studies highlight a critical role for calcium signaling in the regulation of erythroid enucleation.

A Chemical Screening Approach to Identify Novel Key Mediators of Erythroid Enucleation.

Erythroid enucleation is critical for terminal differentiation of red blood cells, and involves extrusion of the nucleus by orthochromatic erythroblasts to produce reticulocytes. Due to the difficulty of synchronizing erythroblasts, the molecular mechanisms underlying the enucleation process remain poorly understood. To elucidate the cellular program governing enucleation, we utilized a novel chemical screening approach whereby orthochromatic cells primed for enucleation were enriched ex vivo and subjected to a functional drug screen using a 324 compound library consisting of structurally diverse, medicinally active and cell permeable drugs. Using this approach, we have confirmed the role of HDACs, proteasomal regulators and MAPK in erythroid enucleation and introduce a new role for Cyclin-dependent kinases, in particular CDK9, in this process. Importantly, we demonstrate that when coupled with imaging analysis, this approach provides a powerful means to identify and characterize rate limiting steps involved in the erythroid enucleation process.

Cell-type-specific transcriptional profiles of the dimorphic pathogen Penicillium marneffei reflect distinct reproductive, morphological, and environmental demands.

Penicillium marneffei is an opportunistic human pathogen endemic to Southeast Asia. At 25° P. marneffei grows in a filamentous hyphal form and can undergo asexual development (conidiation) to produce spores (conidia), the infectious agent. At 37° P. marneffei grows in the pathogenic yeast cell form that replicates by fission. Switching between these growth forms, known as dimorphic switching, is dependent on temperature. To understand the process of dimorphic switching and the physiological capacity of the different cell types, two microarray-based profiling experiments covering approximately 42% of the genome were performed. The first experiment compared cells from the hyphal, yeast, and conidiation phases to identify "phase or cell-state-specific" gene expression. The second experiment examined gene expression during the dimorphic switch from one morphological state to another. The data identified a variety of differentially expressed genes that have been organized into metabolic clusters based on predicted function and expression patterns. In particular, C-14 sterol reductase-encoding gene ergM of the ergosterol biosynthesis pathway showed high-level expression throughout yeast morphogenesis compared to hyphal. Deletion of ergM resulted in severe growth defects with increased sensitivity to azole-type antifungal agents but not amphotericin B. The data defined gene classes based on spatio-temporal expression such as those expressed early in the dimorphic switch but not in the terminal cell types and those expressed late. Such classifications have been helpful in linking a given gene of interest to its expression pattern throughout the P. marneffei dimorphic life cycle and its likely role in pathogenicity.

PhagoSight: an open-source MATLAB® package for the analysis of fluorescent neutrophil and macrophage migration in a zebrafish model.

Neutrophil migration in zebrafish larvae is increasingly used as a model to study the response of these leukocytes to different determinants of the cellular inflammatory response. However, it remains challenging to extract comprehensive information describing the behaviour of neutrophils from the multi-dimensional data sets acquired with widefield or confocal microscopes. Here, we describe PhagoSight, an open-source software package for the segmentation, tracking and visualisation of migrating phagocytes in three dimensions. The algorithms in PhagoSight extract a large number of measurements that summarise the behaviour of neutrophils, but that could potentially be applied to any moving fluorescent cells. To derive a useful panel of variables quantifying aspects of neutrophil migratory behaviour, and to demonstrate the utility of PhagoSight, we evaluated changes in the volume of migrating neutrophils. Cell volume increased as neutrophils migrated towards the wound region of injured zebrafish. PhagoSight is openly available as MATLAB® m-files under the GNU General Public License. Synthetic data sets and a comprehensive user manual are available from http://www.phagosight.org.

HyPer-3: a genetically encoded H(2)O(2) probe with improved performance for ratiometric and fluorescence lifetime imaging.

High-performance sensors for reactive oxygen species are instrumental to monitor dynamic events in cells and organisms. Here, we present HyPer-3, a genetically encoded fluorescent indicator for intracellular H2O2 exhibiting improved performance with respect to response time and speed. HyPer-3 has an expanded dynamic range compared to HyPer and significantly faster oxidation/reduction dynamics compared to HyPer-2. We demonstrate this performance by in vivo imaging of tissue-scale H2O2 gradients in zebrafish larvae. Moreover, HyPer-3 was successfully employed for single-wavelength fluorescent lifetime imaging of H2O2 levels both in vitro and in vivo.

Neutrophil-delivered myeloperoxidase dampens the hydrogen peroxide burst after tissue wounding in zebrafish.

Prompt neutrophil arrival is critical for host defense immediately after injury [1-3]. Following wounding, a hydrogen peroxide (H(2)O(2)) burst generated in injured tissues is the earliest known leukocyte chemoattractant [4]. Generating this tissue-scale H(2)O(2) gradient uses dual oxidase [4] and neutrophils sense H(2)O(2) by a mechanism involving the LYN Src-family kinase [5], but the molecular mechanisms responsible for H(2)O(2) clearance are unknown [6]. Neutrophils carry abundant amounts of myeloperoxidase, an enzyme catalyzing an H(2)O(2)-consuming reaction [7, 8]. We hypothesized that this neutrophil-delivered myeloperoxidase downregulates the high tissue H(2)O(2) concentrations that follow wounding. This was tested in zebrafish using simultaneous fluorophore-based imaging of H(2)O(2) concentrations and leukocytes [4, 9-11] and a new neutrophil-replete but myeloperoxidase-deficient mutant (durif). Leukocyte-depleted zebrafish had an abnormally sustained wound H(2)O(2) burst, indicating that leukocytes themselves were required for H(2)O(2) downregulation. Myeloperoxidase-deficient zebrafish also had abnormally sustained high wound H(2)O(2) concentrations despite similar numbers of arriving neutrophils. A local H(2)O(2)/myeloperoxidase interaction within wound-recruited neutrophils was demonstrated. These data demonstrate that leukocyte-delivered myeloperoxidase cell-autonomously downregulates tissue-generated wound H(2)O(2) gradients in vivo, defining a new requirement for myeloperoxidase during inflammation. Durif provides a new animal model of myeloperoxidase deficiency closely phenocopying the prevalent human disorder [7, 12, 13], offering unique possibilities for investigating its clinical consequences.

In vivo real-time visualization of leukocytes and intracellular hydrogen peroxide levels during a zebrafish acute inflammation assay.

Following injury, the inflammatory response directs the host immune cells to the wound to maintain tissue integrity and combat pathogens. The recruitment of immune cells to inflammatory sites is achieved through the establishment of a variety of signal gradients. Using a zebrafish embryo injury model, it was recently demonstrated that, upon injury, cells at the wound margin rapidly produce hydrogen peroxide (H(2)O(2)) which serves as an early paracrine signal to leukocytes. This chapter provides a method for performing in vivo time-lapse fluorescence microscopy to visualize leukocyte behaviors and wound-produced H(2)O(2) simultaneously in single zebrafish embryos during an acute inflammatory response. Protocols are included for inducing a robust, reproducible acute inflammatory response, for rapidly mounting immobilized embryos for time-lapse imaging, and for computing ratiometric data from the images of embryos expressing the genetically encoded H(2)O(2) sensor fluorophore HyPer. General issues to consider when designing multichannel fluorescent imaging are discussed, including particular considerations to note when monitoring intracellular H(2)O(2) concentration dynamics using HyPer.

Midbrain-hindbrain boundary patterning and morphogenesis are regulated by diverse grainy head-like 2-dependent pathways.

The isthmic organiser located at the midbrain-hindbrain boundary (MHB) is the crucial developmental signalling centre responsible for patterning mesencephalic and metencephalic regions of the vertebrate brain. Formation and maintenance of the MHB is characterised by a hierarchical program of gene expression initiated by fibroblast growth factor 8 (Fgf8), coupled with cellular morphogenesis, culminating in the formation of the tectal-isthmo-cerebellar structures. Here, we show in zebrafish that one orthologue of the transcription factor grainy head-like 2 (Grhl2), zebrafish grhl2b plays a central role in both MHB maintenance and folding by regulating two distinct, non-linear pathways. Loss of grhl2b expression induces neural apoptosis and extinction of MHB markers, which are rescued by re-expression of engrailed 2a (eng2a), an evolutionarily conserved target of the Grhl family. Co-injection of sub-phenotypic doses of grhl2b and eng2a morpholinos reproduces the apoptosis and MHB marker loss, but fails to substantially disrupt formation of the isthmic constriction. By contrast, a novel direct grhl2b target, spec1, identified by phylogenetic analysis and confirmed by ChIP, functionally cooperates with grhl2b to induce MHB morphogenesis, but plays no role in apoptosis or maintenance of MHB markers. Collectively, these data show that MHB maintenance and morphogenesis are dissociable events regulated by grhl2b through diverse transcriptional targets.

Discerning different in vivo roles of microRNAs by experimental approaches in zebrafish.

MicroRNAs (miRNAs) are small endogenous RNAs of approximately 23 nucleotides that regulate the cellular transcriptome by binding to target mRNAs in a sequence-restricted manner, thereby modulating target transcript translation and turnover. Although the direct repressive trans-acting action of miRNAs is to cause a net reduction in the total amount of protein generated from their target mRNAs, developmental and physiological processes have combined this with the flexibility of spatial and temporal regulation of both the miRNAs and their targets to employ miRNAs in a range of regulatory roles. These different roles achieve diverse regulatory outcomes. Five common in vivo regulatory roles of miRNAs are summarized, along with their key defining attributes that could be experimentally addressed to distinguish between them. Methods utilizing zebrafish that are suitable for determining the functional role of a particular miRNA of interest are outlined.

mpeg1 promoter transgenes direct macrophage-lineage expression in zebrafish.

Macrophages and neutrophils play important roles during the innate immune response, phagocytosing invading microbes and delivering antimicrobial compounds to the site of injury. Functional analyses of the cellular innate immune response in zebrafish infection/inflammation models have been aided by transgenic lines with fluorophore-marked neutrophils. However, it has not been possible to study macrophage behaviors and neutrophil/macrophage interactions in vivo directly because there has been no macrophage-only reporter line. To remove this roadblock, a macrophage-specific marker was identified (mpeg1) and its promoter used in mpeg1-driven transgenes. mpeg1-driven transgenes are expressed in macrophage-lineage cells that do not express neutrophil-marking transgenes. Using these lines, the different dynamic behaviors of neutrophils and macrophages after wounding were compared side-by-side in compound transgenics. Macrophage/neutrophil interactions, such as phagocytosis of senescent neutrophils, were readily observed in real time. These zebrafish transgenes provide a new resource that will contribute to the fields of inflammation, infection, and leukocyte biology.

Validating microRNA Target Transcripts Using Zebrafish Assays.

Hundreds of tiny noncoding RNAs known as microRNAs (miRNAs) have been identified in the genomes of plants and animals. Studies are increasingly demonstrating that individual miRNAs are important in normal development and physiology. miRNAs are regulators of gene expression that bind target mRNAs and modulate their translation and turnover. The specificity of this regulation is achieved by partial sequence complementarity between the miRNA and its target mRNA. Understanding which mRNAs are targeted by each particular microRNA is critical to an understanding of the biologic role of any particular miRNA. Bioinformatic approaches can be used to predict mRNAs that may be miRNA targets, but each of these predictions requires experimental validation. We describe a method for a reporter assay based on a fluorescence intensity readout that uses transient techniques in zebrafish to easily deliver the reporter assay components. In addition, we describe a rigorously controlled strategy for determining the bona fide miRNA binding sites in the 3'UTR of mRNAs.

miR-451 regulates zebrafish erythroid maturation in vivo via its target gata2.

We demonstrate that in zebrafish, the microRNA miR-451 plays a crucial role in promoting erythroid maturation, in part via its target transcript gata2. Zebrafish miR-144 and miR-451 are processed from a single precursor transcript selectively expressed in erythrocytes. In contrast to other hematopoietic mutants, the zebrafish mutant meunier (mnr) showed intact erythroid specification but diminished miR-144/451 expression. Although erythropoiesis initiated normally in mnr, erythrocyte maturation was morphologically retarded. Morpholino knockdown of miR-451 increased erythrocyte immaturity in wild-type embryos, and miR-451 RNA duplexes partially rescued erythroid maturation in mnr, demonstrating a requirement and role for miR-451 in erythrocyte maturation. mnr provided a selectively miR-144/451-deficient background, facilitating studies to discern miRNA function and validate candidate targets. Among computer-predicted miR-451 targets potentially mediating these biologic effects, the pro-stem cell transcription factor gata2 was an attractive candidate. In vivo reporter assays validated the predicted miR-451/gata2-3'UTR interaction, gata2 down-regulation was delayed in miR-451-knockdown and mnr embryos, and gata2 knockdown partially restored erythroid maturation in mnr, collectively confirming gata2 down-regulation as pivotal for miR-451-driven erythroid maturation. These studies define a new genetic pathway promoting erythroid maturation (mnr/miR-451/gata2) and provide a rare example of partial rescue of a mutant phenotype solely by miRNA overexpression.

Characterisation of duplicate zinc finger like 2 erythroid precursor genes in zebrafish.

In separate expression pattern and micro-array screens the zinc finger containing factor, znfl2, has been previously implicated in hematopoiesis. Here we analysed znfl2 expression in detail and performed genetic epistatic analysis in a series of hematopoietic mutants and transient gain-of-function models. znfl2 expression in the hematopoietic intermediate mesoderm and derived erythrocytes required early genes cloche and spadetail, but not gata1. Expression was up-regulated in scl gain-of-function embryos, identifying znfl2 as an early erythroid factor that is regulated upstream or independently of gata1. Furthermore, we identified a duplicate znfl2 gene in the genome (znfl2b) which was expressed in early mesendoderm and weakly in the lateral plate mesoderm, overlapping in expression with znfl2. The production of loss-of-function models for znfl2, znfl2b and znfl2/znfl2b together suggested that these erythrocyte specific zinc finger genes are dispensible for erythropoiesis.

Copper stimulates trafficking of a distinct pool of the Menkes copper ATPase (ATP7A) to the plasma membrane and diverts it into a rapid recycling pool.

MNK (Menkes copper-translocating P-type ATPase, or the Menkes protein; ATP7A) plays a key role in regulating copper homoeostasis in humans. MNK has been shown to have a dual role in the cell: it delivers copper to cuproenzymes in the Golgi compartment and effluxes excess copper from the cell. These roles can be achieved through copper-regulated trafficking of MNK. It has previously been shown to undergo trafficking from the trans -Golgi network to the plasma membrane in response to elevated copper concentrations, and to be endocytosed from the plasma membrane to the trans -Golgi network upon the removal of elevated copper. However, the fundamental question as to whether copper influences trafficking of MNK to or from the plasma membrane remained unanswered. In this study we utilized various methods of cell-surface biotinylation to attempt to resolve this issue. These studies suggest that copper induces trafficking of MNK to the plasma membrane but does not affect its rate of internalization from the plasma membrane. We also found that only a specific pool of MNK can traffic to the plasma membrane in response to elevated copper. Significantly, copper appeared to divert MNK into a fast-recycling pool and prevented it from recycling to the Golgi compartment, thus maintaining a high level of MNK in the proximity of the plasma membrane. These findings shed new light on the cell biology of MNK and the mechanism of copper homoeostasis in general.