Hemocyte differentiation to the megacyte lineage enhances mosquito immunity against Plasmodium.

Activation of Toll signaling in Anopheles gambiae by silencing Cactus, a suppressor of this pathway, enhances local release of hemocyte-derived microvesicles (HdMv), promoting activation of the mosquito complement-like system, which eliminates Plasmodium ookinetes. We uncovered the mechanism of this immune enhancement. Cactus silencing triggers a Rel1-mediated differentiation of granulocytes to the megacyte lineage, a new subpopulation of giant cells, resulting in a dramatic increase in the proportion of circulating megacytes. Megacytes are very plastic cells that are massively recruited to the basal midgut surface in response to Plasmodium infection. We show that Toll signaling modulates hemocyte differentiation and that megacyte recruitment to the midgut greatly enhances mosquito immunity against Plasmodium.

Malaria causes hundreds of thousands of deaths each year. This devastating disease is caused by Plasmodium parasites, which are transmitted to people through female Anopheles gambiae mosquitos. Mosquitos become infected with Plasmodium when they ingest blood containing these malaria-causing parasites. However, Plasmodium must avoid the mosquito immune system to survive and spread. The mosquito immune system is made up of several types of immune cells, including cells known as granulocytes. Granulocytes can further develop into additional cell subtypes, such as megacytes and antimicrobial granulocytes, but it is not clear how these types of cells work to protect mosquitos against infections. In the mosquitos that transmit malaria, a cell signaling pathway called Toll helps control immune responses to disease-causing microbes, such as Plasmodium. When Toll signaling is strongly triggered in mosquitos, Plasmodium infection is eliminated because immune cell responses are enhanced ­ which results in lower levels of transmission to humans. But what is the underlying mechanism through which high levels of Toll signaling eradicate Plasmodium infection? To find out, Barletta et al. collected cell samples from A. gambiae mosquitos and analyzed what happened when Toll signaling was strongly activated. They observed a large increase in the proportion of megacytes in these mosquitos (from 2% to 80% of all granulocytes). Toll signaling also caused megacytes to become bigger, cluster together, and have higher plasticity ­ meaning they could adopt different shapes. Barletta et al. used microscopy to show that these megacytes were releasing large mitochondria-like structures and membrane vesicles , which may be the trigger activating the mosquito's immune system. In live mosquitos, megacytes move towards the area of the Plasmodium infection and release microvesicles. These microvesicles are known to activate a part of the the mosquito's immune system called the complement-like system, destroying the parasites and preventing mosquito infection and disease transmission. These findings show how strong Toll signaling triggers the mosquito immune system to eliminate Plasmodium infections. Understanding how the mosquito immune system tackles Plasmodium infection may help reveal ways to reduce or block transmission.

Live In Vivo Imaging of Plasmodium Invasion of the Mosquito Midgut.

The mosquito midgut is a critical barrier that Plasmodium parasites must overcome to complete their developmental cycle and be transmitted to a new vertebrate host. Previous confocal studies with fixed infected midguts showed that ookinetes traverse midgut epithelial cells and cause irreversible tissue damage. Here, we investigated the spatiotemporal dynamics of ookinete midgut traversal and the response of midgut cells to invasion. A novel mounting strategy was established, suitable fluorescent dye combinations were identified and protocols optimized to label mosquito tissues in vivo, and live imaging protocols using confocal microscopy were developed. Tracking data showed that ookinetes gliding on the midgut surface travel faster and farther than those that remain in the lumen or those that have invaded the epithelium. Image analysis confirmed that parasite invasion and cell traversal occur within a couple of minutes, while caspase activity in damaged cells, indicative of cellular apoptosis, and F-actin cytoskeletal rearrangements in cells extruded into the gut lumen persist for several hours. This temporal difference highlights the importance of hemocyte-mediated cellular immunity and the mosquito complement system to mount a coordinated and effective antiplasmodial response. This novel in vivo imaging protocol allowed us to continuously observe individual ookinetes in live mosquitoes within the gut lumen and during cell traversal and to capture the subsequent cellular responses to invasion in real time for several hours, without loss of tissue integrity.IMPORTANCE Malaria is one of the most devastating parasitic diseases in humans and is transmitted by anopheline mosquitoes. The mosquito midgut is a critical barrier that Plasmodium parasites must overcome to complete their developmental cycle and be transmitted to a new host. Here, we developed a new strategy to visualize Plasmodium ookinetes as they traverse the mosquito midgut and to follow the response of damaged epithelial cells by imaging live mosquitoes. Understanding the spatial and temporal aspects of these interactions is critical when developing novel strategies to disrupt disease transmission.

Mosquito Midgut Prostaglandin Release Establishes Systemic Immune Priming.

Anopheles gambiae mosquitoes that have been infected with Plasmodium mount a more effective immune response to a subsequent infection. Priming is established when Plasmodium invasion of the mosquito midgut allows contact of the gut microbiota with epithelial cells. This event is followed by a systemic release of a hemocyte differentiation factor (HDF) consisting of Lipoxin A4 bound to Evokin, a lipocalin carrier, which increases the proportion of circulating hemocytes. We show that mosquito midgut cells produce and release prostaglandin E2 (PGE2), which attracts hemocytes to the midgut surface and enhances their patrolling activity. Systemic injection of prostaglandins (PGs) recapitulates the priming response and enhances antiplasmodial immunity by triggering HDF production. Although insects lack cyclooxygenases, two heme peroxidases, HPX7 and HPX8, catalyze essential steps in PG biosynthesis in mosquitoes. Mosquito midgut PGE2 release attracts hemocytes and establishes a long-lasting enhanced systemic cellular immune response to Plasmodium infection.

Activation of mosquito complement antiplasmodial response requires cellular immunity.

The mosquito complement-like system is a major defense mechanism that limits Plasmodium infection. Ookinete midgut invasion results in irreversible damage to invaded cells and triggers epithelial nitration and complement activation. Several lines of evidence suggest that hemocytes participate in early antiplasmodial responses that target ookinetes, but their role remains unclear. The fate of hemocytes in response to Plasmodium infection was investigated by labeling this cell population in vivo. We found that midgut nitration triggers the local release of hemocyte-derived microvesicles (HdMv) into the basal labyrinth of the midgut. Several different strategies, such as gene silencing, immune priming, or systemic injection of polystyrene beads, were used to either enhance or reduce HdMv release. We provide direct experimental evidence that contact of hemocytes with the nitrated midgut basal surface triggers HdMv release and that this response is necessary for effective activation of mosquito complement. Our studies suggest that hemocyte-derived microvesicles may deliver some critical factor(s) that promote activation of thioester-containing protein 1, a key effector of the mosquito antiplasmodial immunity.

The migrations of Drosophila muscle founders and primordial germ cells are interdependent.

Caudal visceral mesoderm (CVM) cells migrate from posterior to anterior of the Drosophila embryo as two bilateral streams of cells to support the specification of longitudinal muscles along the midgut. To accomplish this long-distance migration, CVM cells receive input from their environment, but little is known about how this collective cell migration is regulated. In a screen we found that wunen mutants exhibit CVM cell migration defects. Wunens are lipid phosphate phosphatases known to regulate the directional migration of primordial germ cells (PGCs). PGC and CVM cell types interact while PGCs are en route to the somatic gonadal mesoderm, and previous studies have shown that CVM impacts PGC migration. In turn, we found here that CVM cells exhibit an affinity for PGCs, localizing to the position of PGCs whether mislocalized or trapped in the endoderm. In the absence of PGCs, CVM cells exhibit subtle changes, including more cohesive movement of the migrating collective, and an increased number of longitudinal muscles is found at anterior sections of the larval midgut. These data demonstrate that PGC and CVM cell migrations are interdependent and suggest that distinct migrating cell types can coordinately influence each other to promote effective cell migration during development.

Ectopic expression screen identifies genes affecting Drosophila mesoderm development including the HSPG Trol.

Gastrulation of the embryo involves coordinate cell movements likely supported by multiple signaling pathways, adhesion molecules, and extracellular matrix components. Fibroblast growth factors (FGFs) have a major role in Drosophila melanogaster mesoderm migration; however, few other inputs are known and the mechanism supporting cell movement is unclear. To provide insight, we performed an ectopic expression screen to identify secreted or membrane-associated molecules that act to support mesoderm migration. Twenty-four UAS insertions were identified that cause lethality when expressed in either the mesoderm (Twi-Gal4) or the ectoderm (69B-Gal4). The list was narrowed to a subset of 10 genes that were shown to exhibit loss-of-function mutant phenotypes specifically affecting mesoderm migration. These include the FGF ligand Pyramus, α-integrins, E-cadherin, Cueball, EGFR, JAK/STAT signaling components, as well as the heparan sulfate proteoglycan (HSPG) Terribly reduced optic lobes (Trol). Trol encodes the ortholog of mammalian HSPG Perlecan, a demonstrated FGF signaling cofactor. Here, we examine the role of Trol in Drosophila mesoderm migration and compare and contrast its role with that of Syndecan (Sdc), another HSPG previously implicated in this process. Embryos mutant for Trol or Sdc were obtained and analyzed. Our data support the view that both HSPGs function to support FGF-dependent processes in the early embryo as they share phenotypes with FGF mutants: Trol in terms of effects on mesoderm migration and caudal visceral mesoderm (CVM) migration and Sdc in terms of dorsal mesoderm specification. The differential roles uncovered for these two HSPGs suggest that HSPG cofactor choice may modify FGF-signaling outputs.

Image analysis and empirical modeling of gene and protein expression.

Protein gradients and gene expression patterns are major determinants in the differentiation and fate map of the developing embryo. Here we discuss computational methods to quantitatively measure the positions of gene expression domains and the gradients of protein expression along the dorsal-ventral axis in the Drosophila embryo. Our methodology involves three layers of data. The first layer, or the primary data, consists of z-stack confocal images of embryos processed by in situ hybridization and/or antibody stainings. The secondary data are relationships between location, usually an x-axis coordinate, and fluorescent intensity of gene or protein detection. Tertiary data comprise the optimal parameters that arise from fits of the secondary data to empirical models. The tertiary data are useful to distill large datasets of imaged embryos down to a tractable number of conceptually useful parameters. This analysis allows us to detect subtle phenotypes and is adaptable to any set of genes or proteins with a canonical pattern. For example, we show how insights into the Dorsal transcription factor protein gradient and its target gene ventral-neuroblasts defective (vnd) were obtained using such quantitative approaches.

The role of FGF signaling in guiding coordinate movement of cell groups: guidance cue and cell adhesion regulator?

Cell migration influences cell-cell interactions to drive cell differentiation and organogenesis. To support proper development, cell migration must be regulated both temporally and spatially. Mesoderm cell migration in the Drosophila embryo serves as an excellent model system to study how cell migration is controlled and influences organogenesis. First, mesoderm spreading transforms the embryo into a multilayered form during gastrulation and, subsequently, cells originating from the caudal visceral mesoderm (CVM) migrate along the entire length of the gut. Here we review our studies, which have focused on the role of fibroblast growth factor (FGF) signaling, and compare and contrast these two different cell migration processes: mesoderm spreading and CVM migration. In both cases, FGF acts as a chemoattractant to guide cells' directional movement but is likely not the only signal that serves this role. Furthermore, FGF likely modulates cell adhesion properties since FGF mutant phenotypes share similarities with those of cell adhesion molecules. Our working hypothesis is that levels of FGF signaling differentially influence cells' response to result in either directional movement or changes in adhesive properties.

Dorsal-ventral gene expression in the Drosophila embryo reflects the dynamics and precision of the dorsal nuclear gradient.

Patterning of the dorsal-ventral axis in the early Drosophila embryo depends on the nuclear distribution of the Dorsal transcription factor. Using live two-photon light-sheet microscopy, we quantified the nuclear Dorsal gradient in space and time and found that its amplitude and basal levels display oscillations throughout early embryonic development. These dynamics raise questions regarding how cells can reproducibly establish patterns of gene expression from a rapidly varying signal. We therefore quantified domains of Dorsal target genes, discovering their expression patterns are also dynamic. Computational modeling of this system reveals a correlation between Dorsal gradient dynamics and changes in target gene expression and suggests that these dynamics, together with time averaging of noise, results in the formation of graded gene expression borders in regions where the gradient is nearly flat. We propose that mRNA levels remain plastic during transient signaling events, allowing tissues to refine patterns in the face of genetic or environmental variation.