MF Portereiko et al. (1999) International C. elegans Meeting "Pharyngeal Morphogenesis"

MF Portereiko, ME Domeier, SE Mango

[International C. elegans Meeting, 1999]

Our lab is interested in studying pharyngeal morphogenesis, the process whereby cells of the pharyngeal primordium elongate to form a tube that connects to the buccal cavity. We have taken two approaches towards understanding this process. First, we analyzed the movements associated with wildtype pharyngeal morphogenesis. We built a chimera composed of a digestive tract-specific promoter controlling GFP expression, and we targeted GFP to the plasma membrane by including the 'CAAX' sequence from mig-2 1 . We introduced the GFP chimera into wildtype animals and observed the behavior of pharyngeal cells using time-lapse microscopy 2 . Our preliminary studies suggest that pharyngeal morphogenesis depends in large part on directed cell shape changes, and not on intercalation (e.g. convergence/extension) nor cellular migration. The cumulative effects of anterior pharyngeal cells increasing their longitudinal dimension appears to be sufficient to bridge the distance between the pharyngeal primoridum and the nascent buccal cavity. These data suggest that directed changes in the actin cytoskeleton may be important for pharyngeal elongation. Second, we searched for loci involved in pharyngeal morphogenesis using a deficiency screen. We surveyed approximately a third of the genome for deficiencies with pharyngeal elongation defects and chose two for further analysis. i) mnDf90 embryos form a pharyngeal primordium that fails to attach to the buccal cavity (see abstract by Lange and Mango); ii) uDf1 embryos attach to the buccal cavity similar to wildtype embryos. However, these mutants form a bifurcated tube with two lumens 3 . This phenotype could reflect a defect in cellular positioning within the primordium. Alternatively, the cell shape or polarity changes that normally occur during morphogenesis may be disrupted. These embryos contain the wildtype number of pharyngeal cells, and appear to differentiate normally. Our current goals are to follow cellular behaviors in the deficiency embryos as well as identify single point mutations that mimic the deficiency phenotypes. 1. Zipkin et al., 1997 2. Special thanks to Bill Mohler, John White and the IMR for their help. 3. see also Terns et al., 1997

Portereiko MF et al. (2000) West Coast Worm Meeting "Pharyngeal extension: the short and the long of it"

Mango SE, Portereiko MF

[West Coast Worm Meeting, 2000]

Pharyngeal morphogenesis initiates during mid-embryogenesis, when 78 of the 80 pharyngeal cells have been born, and the embryo has begun to elongate. At this time, the pharyngeal precursors form a compact ball that is attached to the nascent midgut and surrounded by a basement membrane that encloses the entire primordium except for a gap at the anterior. Over the next 80 minutes, the pharyngeal precursors alter their morphology and position to form a linear tube that is linked to the buccal cavity anteriorly and the midgut posteriorly. We call this process pharyngeal extension. We have used time-lapse videomicroscopy and GFP reporter constructs to follow the behavior of the pharyngeal precursors during pharyngeal extension. Our studies demonstrate that pharyngeal extension can be loosely divided into three stages i) reorganization of cellular polarity within pharyngeal cells (specifically the pharyngeal epithelial precursors), ii) formation of an epithelium that mechanically couples the pharyngeal cells to arcade cells in the nascent buccal cavity, and iii) an apparent contraction that shifts the buccal cavity posteriorly and the pharynx anteriorly. We are considering a 'purse string' model to explain the cellular behaviors we see during contraction. Our findings suggest that pharyngeal extension is the result of 'pulling' by anterior pharyngeal cells rather than 'pushing' by posterior pharyngeal cells. To test this idea, we eliminated the posterior pharynx via genetic and physical means and demonstrated that pharyngeal extension still occurs normally. Our model also predicts that tension between cells of the pharynx and buccal cavity is required for the movement we observe during contraction. We tested this idea by destroying the arcade cells. As expected, this manipulation blocked the anterior-directed movement of the pharyngeal cells and reduced the posterior-directed movement of the buccal cavity. Our current goal is to identify molecules required for pharyngeal extension using forward and reverse genetic approaches.

Portereiko MF et al. (2001) Dev Biol "Early morphogenesis of the Caenorhabditis elegans pharynx."

Mango SE, Portereiko MF

[Dev Biol, 2001]

We investigated the cellular behaviors that accompany the early stages of pharyngeal morphogenesis in Caenorhabditis elegans. The embryonic pharynx develops from a ball of cells into a linear tube connected anteriorly to the buccal cavity and posteriorly to the midgut. By using GFP reporters localized to discrete subcellular regions, we show that pharyngeal morphogenesis can be divided into three stages: (1) lengthening of the nascent pharyngeal lumen by reorientation of apicobasal polarity of anterior pharyngeal cells ("Reorientation"), (2) formation of an epithelium by the buccal cavity cells, which mechanically couples the buccal cavity to the pharynx and anterior epidermis ("Epithelialization"), and (3) a concomitant movement of the pharynx anteriorly and the epidermis of the mouth posteriorly to bring the pharynx, buccal cavity, and mouth into close apposition ("Contraction"). Several models can account for these cellular behaviors, and we distinguish between them by physically or genetically ablating cells within the digestive tract. These studies provide the first description of how the pharynx primordium develops into an epithelial tube, and reveal that pharyngeal morphogenesis resembles aspects of mammalian kidney tubulogenesis.

Portereiko MF et al. (2004) Curr Biol "ZEN-4/MKLP1 is required to polarize the foregut epithelium."

Mango SE, Saam J, Portereiko MF

[Curr Biol, 2004]

Background: Epithelial tubes are a key component of organs and are generated from cells with distinct apico-basolateral polarity. Here, we describe a novel function during tubulogenesis for ZEN-4, the Caenorhabditis elegans ortholog of mitotic kinesin-like protein 1 (MKLP1), and CYK-4, which contains a RhoGAP (GTPase-activating protein) domain. Previous studies revealed that these proteins comprise centralspindlin (a complex that functions during mitosis to bundle microtubules), construct the spindle midzone, and complete cytokinesis. Results: Our analyses demonstrate that ZEN-4/MKLP1 functions postmitotically to establish the foregut epithelium. Mutants that lack ZEN-4/MKLP1 express polarity markers but fail to target these proteins appropriately to the cell cortex. Affected proteins include PAR-3/Bazooka and PKC-3/atypical protein kinase C at the apical membrane domain, and HMR-1/cadherin and AJM-1 within C. elegans apical junctions (CeAJ). Microtubules and actin are disorganized in zen-4 mutants compared to the wild-type. Conclusion: We suggest that ZEN-4/MKLP1 and CYK-4/RhoGAP regulate an early step in epithelial polarization that is required to establish the apical domain and CeAJ.

Portereiko MF et al. (1998) West Coast Worm Meeting "IDENTIFICATION OF ZYGOTIC LOCI THAT PATTERN THE PHARYNX PRIMORDIUM"

Mango SE, Portereiko MF

[West Coast Worm Meeting, 1998]

During pharyngeal development, the pharyngeal precursor cells cluster together to form a primordium that subsequently undergoes differentiation and morphogenesis. We have taken a deficiency approach to identify genes that pattern the pharynx primordium. Previous studies from the Fire, Rothman, Wood and Labouesse laboratories have described the phenotypes associated with deficiencies that collectively delete >75% of the genome1. On the basis of their initial characterization we have focused on eleven deficiencies that appear likely to have specific defects in pharynx development. Using Nomarski DIC microscopy and antibody staining, we have classified the eleven deficiencies into three phenotypic groups. The first group of deficiencies gives rise to a pharynx primordium that fails to differentiate. In one deficiency, this defect may reflect a problem in the formation or maintenance of epithelia throughout the embryo, since midgut and hypodermal cells are also affected. The second group generates small pharynges. Fewer pharyngeal cells appear to be produced. Alternatively, there may be defects in the growth or morphogenesis of individual pharyngeal cells. The third group of deficiencies produces a bifurcated pharynx. Instead of forming a linear tube, the pharyngeal lumen branches at a reproducible location along the pharyngeal axis. We have focused on this last class of deficiency. Three explanations could account for the bifurcation phenotype. First, cells that would normally choose a non-pharyngeal fate might adopt a pharyngeal fate instead. To test this hypothesis, we counted the number of PHA-4+ cells in mutant embryos. PHA-4 is expressed in all pharyngeal cells in wildtype embryos, and is therefore a useful marker for pharyngeal fate. None of the three bifurcation deficiencies produced extra numbers of PHA-4+ cells, arguing against the first hypothesis. Second, these loci could affect cell fate specification within the pharynx primordium. Third, there might be a defect in the morphogenesis of the primordium from a ball of cells into a linear tube. Our current goal is to identify the genes responsible for the bifurcated phenotype. Towards this aim we are characterizing the phenotype of smaller deficiencies in the three regions and we are screening for EMS-induced mutations that give rise to the bifurcated phenotype. 1. Ahnn and Fire, 1994; Storfer-Glazer and Wood, 1994; Chanal and Labouesse, 1997; Labouesse, 1997; Terns et al., 1997,

Lawrence RA et al. (2000) Parasitology "Requirements for in vivo IFN-gamma induction by live microfilariae of the parasitic nematode, Brugia malayi."

Allen JE, Gray CA, Lawrence RA

[Parasitology, 2000]

Lymphatic filariasis caused by the parasitic nematode, Brugia malayi, is a chronic human disease immunologically characterized by stimulation of Th2 cells and reduced antigen-specific T cell responses. Single stage intra-peritoneal infections with infective larvae (L3) or adult nematodes induce Th2 cells, while the microfilarial stage (Mf) stimulates IFN-gamma and Mf-specific IgG1, IgG2a, IgG2b, IgG3 and IgM, but not IgE. To investigate whether IFN-gamma is elicited by live Mf in their natural site of infected, mice were infected intravenously. Intravenous infection had a striking effect on the response to Mf and high levels of IgE were induced even in the presence of IFN-gamma. Indeed IgE levels to Mf increased markedly with the number of immunizations, higher doses of Mf and prolonged exposure to Mf suggesting that under conditions of chronic antigen exposure, typical of human disease, Mf will stimulate high levels of IgE. The ability of Mf-induced IFN-gamma to modulate or regulate a pre-existing Th2 response, was investigated by infecting mice initially with adult male worms to induce a Th2 response, followed 14 days later by infection with Mf. Although Mf stimulated IFN-gamma in the presence of male adults, the antibody isotypes elicited did not reflect IFN-gamma induction and IgG1 and IgE dominated the response. Although it cannot be discounted that IFN-gamma induction by Mf may act locally as an inflammatory mediator or modulator of Th2 cells, these data suggest that Mf-stimulated IFN-gamma does not have a profound effect overall on progression of the Th2-dominated immune response to filarial infection.

Rao UR et al. (1987) Immunol Cell Biol "Complement activation by eggs and microfilariae of filarial parasites."

Rao UR, Subrahmanyam D, Chandrashekar R

[Immunol Cell Biol, 1987]

The complement of fresh normal rat serum was activated by filarial eggs and microfilariae (mf). C3 was deposited on the surface of Litomosoides carinii, Brugia pahangi, Brugia malayi and Dipetalonema viteae as seen by immunofluorescence. Intra-uterine and in vitro-derived mf did not bind C3. In contrast, C3 bound to the blood-derived mf of B. pahangi and B. malayi as well as exsheathed mf of L. carinii and B. malayi. Significant consumption of complement was observed with eggs of all filarial species, as well as sheathed mf of B. pahangi, B. malayi and exsheathed mf of L. carinii and B. malayi. These experiments indicated that complement was activated by filarial parasites via the alternative pathway. The bound complement promoted neutrophil-mediated adherence and cytotoxicity.

Beerntsen BT et al. (2007) J Med Entomol "Response of Armigeres subalbatus (Diptera: Culicidae) to intraperitoneally isolated Brugia spp. microfilariae."

Lowery RJ, Beerntsen BT

[J Med Entomol, 2007]

The relationship between mosquito and parasite involves a delicate balance that is influenced not only by the mosquito but also by parasite determinants. Using the biologically and morphologically similar parasites Brugia malayi and Brugia pahangi and the mosquito Armigeres subalbatus (Coquillett) (Diptera: Culicidae), it should be possible to dissect out the key elements involved in initiating or avoiding an immune response, known as melanotic encapsulation, because in this mosquito B. malayi microfilariae (mf) are melanized and destroyed, but B. pahangi mf develop normally into infective-stage larvae. Because of limitations in isolating sufficient mf from the circulation of an infected mammalian host, Brugia spp. mf that can be obtained in large numbers from the peritoneal cavity of an infected host were tested to ascertain the immune response of Ar. subalbatus to this source of mf. Results indicate that the immune response of Ar. subalbatus against intraperitoneal (i.p.) Brugia spp. mf mimics that which is observed when this mosquito is exposed to mf-infected animals, indicating that i.p. mf are similar to those mf that circulate naturally in the blood of the vertebrate host. Therefore, the i.p. mf should serve as an excellent source of material for genomic and proteomic studies designed to analyze the role of the parasite in influencing the immune response of the mosquito.

Simons JE et al. (2010) Parasite Immunol "Absence of regulatory IL-10 enhances innate protection against filarial parasites by a neutrophil-independent mechanism."

Lawrence RA, Simons JE, Gray CA

[Parasite Immunol, 2010]

Brugia malayi causes the major tropical disease, lymphatic filariasis. Chronicity of disease is associated with generation of regulatory cells secreting IL-10 and/or TGF-beta. Previous work has shown that the rate of microfilariae (Mf) clearance from the blood is mouse strain-dependent. Here, we show that IL-10 plays an important role in preventing the clearance of Mf. Indeed, anti-IL-10 antibody treatment increases the rate of Mf clearance from the bloodstream in both rapid-Mf-clearing CBA/Ca and slow-clearing C57Bl/6 mice. In addition, IL-10(-/-) mice implanted intraperitoneally with Mf-producing adult nematodes have significantly lower Mf, but not adults, in comparison with wild-type mice at 3 weeks post-implantation (p.i.). Clearance of Mf from the peritoneal cavity of IL-10(-/-) mice is associated with a dramatic infiltration of neutrophils. Furthermore, rapid-Mf-clearing CBA/Ca mice have a dramatic blood neutrophilia at 24 h p.i., whereas slow-clearing C57Bl/6 mice show no such neutrophilia. Thus, neutrophils may play a role as effector cells in microfilarial infection. We therefore treated mice with anti-granulocyte antibody to abolish neutrophil recruitment during Mf infection i.v. Although anti-granulocyte treatment severely depleted neutrophils, it did not significantly reduce the rate of B. malayi Mf clearance either during primary infection or during a challenge following antigen sensitization.

Edwards MK et al. (1990) Trop Med Parasitol "Antigenic and dynamic properties of the surface of Onchocerca microfilariae."

Busto P, James ER, Edwards MK, Carlow CK, Philipp M

[Trop Med Parasitol, 1990]

We analyzed the antigenicity and stability of the surface of skin microfilariae (mf) of Onchocerca cervicalis, a horse parasite. These mf express antigens on their surface that are cross-reactive with the cattle parasite O. lienalis and with the human parasite O. volvulus. The surface of living O. cervicalis mf was radioiodinated using Iodogen and the labeled components were solubilized in buffers containing sodium dodecyl sulphate (SDS), or extracted with the milder detergent octyl-beta-D-glucopyranoside (OGP). Electrophoresis of this material showed seven prominent bands, one of which (14 kDa) was specifically precipitated by antisera from rabbits immunized with mf from either O. cervicalis, O. lienalis, or O. volvulus, and by human sera obtained from infected individuals in Chiapas, Mexico. Other components were precipitated by either the rabbit or the human sera. In addition, antisera from mice immunized with O. cervicalis mf bound specifically to the surface of freeze-thawed uterine O. lienalis and O. volvulus mf as detected by immunofluorescence. This fluorescence was lost from the surface of O. cervicalis mf in a temperature-dependent fashion. Live mf incubated on ice with mouse anti-mf antisera and secondary FITC-GAM, showed uniform surface fluorescence. When these mf were incubated at 37 degrees C, but not at 0 degrees C, the fluorescent pattern changed with time. First, small non-fluorescent patches arose, followed by an increasingly wide belt devoid of fluorescence, and finally, no visible fluorescence. These changes in the mf surface suggest potential mechanisms for immune evasion by filarial parasites.