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. (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,

Michael F Portereiko et al. (2001) International C. elegans Meeting "Pharynx tubulogenesis during C. elegans development"

Michael F Portereiko, Susan E Mango

[International C. elegans Meeting, 2001]

During C. elegans embryogenesis, the pharyngeal primordium develops from a ball of cells into a linear tube connected anteriorly to the buccal cavity and posteriorly to the midgut. Using GFP reporters localized to discrete subcellular regions, we have shown that pharyngeal tubulogenesis occurs in three stages: i) lengthening of the nascent pharyngeal lumen by reorientation of apicobasal polarity of anterior pharyngeal cells (Reorientation), ii) formation of an epithelium by the buccal cavity cells, which mechanically couples the buccal cavity to the pharynx and anterior epidermis (Epithelialization), and iii) 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) (1). We call this three-step process pharyngeal extension. We have undertaken two approaches to identify loci required for pharyngeal extension. First, we have used RNA interference to determine the role, if any, of candidate genes previously shown to be expressed in the pharynx. Second, we are undertaking a mutagenesis screen to identify mutants that generate pun (pharynx unattached) phenotypes but are otherwise largely normal. From 2000 haploid genomes, we have recovered seven Pun mutants. Our current goals are to characterize the phenotypes of the mutants and to continue screening. 1. MF Portereiko and SE Mango, Morphogenesis of the C. elegans Pharynx, Dev. Biol, in press.

Glenda Walker et al. (2001) International C. elegans Meeting "Heat Shock Factor in C. elegans and in parasitic nematodes"

Theresa Scanlon, Fiona Thompson, Andrena Brawley, Glenda Walker, Eileen Devaney

[International C. elegans Meeting, 2001]

Filarial parasites of the genus Brugia live in mammals (including humans) and are transmitted between hosts by the bite of a blood-feeding mosquito vector. The infective form for the mosquito is the L1 or microfilariae (Mf) a life cycle stage that is highly adapted for life in the bloodstream of the mammal. Mf are developmentally arrested and undergo no further development until ingested by a mosquito. The link between the progression of the developmental cycle and the transition between hosts implies that the Mf has a mechanism by which it can sense its changing environment. Results in other systems have shown that heat shock factor (HSF) can act as a cellular thermometer directly monitoring temperature and oxidative state. As temperature is one of the major differences between the mosquito and mammalian hosts we are interested in investigating the role of HSF in developmental progression. As the tools for functional genomics do not exist for Brugia, we are using C. elegans as a model system in which to define the role of HSF in a nematode. ACeDB identifies a single HSF-like gene with a highly conserved DNA binding domain. We have used an RNAi feeding vector to determine the likely function of HSF in the nematode. A number of different phenotypes were apparent at 20oC including: slow growth, scrawny appearance, thermo-sensitivity, decreased fertility and egg-laying defective. At 25oC these phenotypes were more pronounced. In an hsp-16/GFP reporter background, RNAi abolished GFP expression in the intestine but not in the pharynx or nerve ring. These results are consistent with a requirement for HSF function in the gut. Ongoing studies are focused on determining the spatial and temporal expression pattern of hsf throughout development. We aim to determine the pathways in which HSF is active under normal developmental conditions and to identify down-stream targets of HSF.

Von Stetina, Stephen E. et al. (2009) International Worm Meeting "Finding Regulators of Cadherin-Independent Epithelialization."

Mango, Susan E., Von Stetina, Stephen E.

[International Worm Meeting, 2009]

How are polarized epithelia established and maintained? This question is of critical importance, as the loss of epithelial polarity is associated with metastasis(1). There are many well-studied protein complexes that lie in specific membrane compartments with roles integral to the epithelial cell. The E-cadherin-containing adherens junction serves to link neighboring epithelial cells together while the more basal tight junction functions to separate the apical and basolateral surfaces. For some cells, E-cadherin is the major initiator of cell polarity and epithelium formation via cell-cell adhesion(2). However, recent studies have discovered E-cadherin independent polarity pathways(3-6). C. elegans offers a powerful system to study this cadherin-independent mechanism, as E-cadherin is dispensible for the initiation of epithelial polarity in nematodes(4). We study cadherin-independent epithelium formation during pharynx development. Nine pharyngeal arcade cells undergo a mesenchymal-to-epithelial transition to link the pharynx to the outer epidermis(7). Ablation of the arcade cells results in a Pharynx unattached (Pun) phenotype, in which the pharynx fails to connect to the epidermis(7). Pun animals die as they are unable to eat. Our lab has undertaken a genetic screen for Pun mutants that fail to form the arcade cell epithelium (Portereiko and Mango, unpublished). This screen revealed that loss of the central-spindlin component ZEN-4/MKLP1 induces a Pun phenotype because the arcade cells fail to polarize(8). We are currently studying where and when ZEN-4 is needed for arcade cell polarization. We have also undertaken a structure/function analysis of this mitotic kinesin in order to elucidate its role in epithelialization. In addition, we are in the process of cloning several mutants that were isolated in the Pun mutagenesis screen. (1). J. M. Lee, S. Dedhar, R. Kalluri, E. W. Thompson, J Cell Biol 172, 973 (Mar 27, 2006). (2). L. N. Nejsum, W. J. Nelson, J Cell Biol 178, 323 (Jul 16, 2007). (3). A. F. Baas et al., Cell 116, 457 (Feb 6, 2004). (4). M. Costa et al., J Cell Biol 141, 297 (Apr 6, 1998). (5). T. J. Harris, M. Peifer, J Cell Biol 167, 135 (Oct 11, 2004). (6). W. B. Raich, C. Agbunag, J. Hardin, Curr Biol 9, 1139 (Oct 21, 1999). (7). M. F. Portereiko, S. E. Mango, Dev Biol 233, 482 (May 15, 2001). (8). M. F. Portereiko, J. Saam, S. E. Mango, Curr Biol 14, 932 (Jun 8, 2004).

Glenda Walker et al. (2002) European Worm Meeting "Heat Shock Factor in Caenorhabditis elegans and in parasitic nematodes"

Glenda Walker, Fiona Thompson, Theresa Scanlon, Andrena Brawley, Eileen Devaney

[European Worm Meeting, 2002]

Filarial parasites of the genus Brugia live in mammals (including humans) and are transmitted between hosts by the bite of a blood-feeding mosquito vector. The infective form for the mosquito is the L1 or microfilariae (Mf) a life cycle stage that is highly adapted for life in the bloodstream of the mammal. Mf are developmentally arrested and undergo no further development until ingested by a mosquito. The link between the progression of the developmental cycle and the transition between hosts implies that the Mf has a mechanism by which it can sense its changing environment. Results in other systems have shown that heat shock factor (HSF) can act as a cellular thermometer directly monitoring temperature and oxidative state. As temperature is one of the major differences between the mosquito and mammalian hosts we are interested in investigating the role of HSF in developmental progression. As the tools for functional genomics do not exist for Brugia, we are using C. elegans as a model system in which to define the role of HSF in a nematode. ACeDB identifies a single HSF-like gene with a highly conserved DNA binding domain. Using an RNAi feeding vector containing fragments of different lengths of C.elegans HSF, we have defined a number of phenotypes. The penetrance of these phenotypes increases with the size of the RNAi fragment and higher growth temperatures. RNAi treated worms have defects in thermotolerance, lifespan, fertility and egg-laying. In addition treated worms are significantly smaller and have a scrawny appearance. In an hsp- 16/GFP reporter background, RNAi abolished GFP expression in the intestine but not in the pharynx or nerve ring. The decrease in HSP-16 levels has been confirmed by immunoblotting. Using a yolk protein/GFP reporter, flourescence in the oocytes is significantly reduced in RNAi treated worms. These results may relate to defects in gut function in RNAi treated worms, and this is consistent with a decreased gut function as defined by feeding assays. Ongoing studies are focused on determining the spatial and temporal expression pattern of hsf throughout development. Our aims are to determine the pathways in which HSF is active under normal developmental conditions and to identify down-stream targets of HSF.

Khan, Liakot et al. (2015) International Worm Meeting "Peri-lumenal intermediate filaments and microtubules maintain a single lumen in extending C. elegans excretory canals."

Gobel, Verena, Khan, Liakot, Membreno, Edward, Zhang, Hongjie

[International Worm Meeting, 2015]

In an RNAi-based modifier screen we identified 3 intermediate filaments (IFs: IFA-4, IFB-1, IFC-2) that enhanced the cystic ERM-1-overexpression excretory canal (EC) phenotype. Although IFB-1's function in EC morphogenesis has been noted, it remains unclear how IFs contribute to this process. Here we compare IFs' role in EC tubulogenesis with the roles of microfilaments (MFs; previously analyzed) and microtubules (MTs). IFs were found to build an intracellular perilumenal lattice, and to be non-redundantly required for lumen extension. Severe interference with IFs causes cell-body close cysts and loss of canal extension, but does not abort lumen formation. In contrast, severe interference with MFs via ERM-1/ACT-5, that we think expand the lumenal membrane via vesicle fusion, abolishes lumen formation. Selective IF removal during larval EC extension generates a "multiple lumen varicosity" phenotype with collapsed lumens between varicosities (periodic structures along extending canals that serve as osmotically sensitive "fueling stations" for wild-type growth). In contrast, larval MF removal produces short, thin lumens with small vacuoles at canal tips. IF removal generates true ectopic "lumens" (vacuoles lined with an apical membrane and cytoskeleton) not seen with MF removal. This scenario could suggest that IFs function by laterally integrating vacuoles into a lumenal membrane that has already acquired an actin coat, as opposed to MF-dependent membrane expansion via vesicle fusion at the tip that concomitantly assembles actin. A TBB-2/tubulin::GFP fusion localizes to the EC cytoplasm, but also to strands on the perilumenal IF lattice that in turn resides on perilumenal MFs. Colocalization and loss-of-function studies during EC development suggest that ERM-1 indirectly recruits lumenal IFs, whereas perilumenal lattice formation may depend on MTs. Interference with MTs copies the IF phenotypes and, unlike interference with MFs, enhances it. Thus, MTs like IFs support single lumen maintenance and lateral vacuole fusion, suggesting that one of MTs' roles in lumen extension is mediated by IFs. Effects of the 3 cytoskeletal components on endosomal and canalicular vesicles will be presented. Our findings suggest that EC lumen extension relies on tip and lateral growth and requires a resilient perilumenal IF matrix that allows vacuoles to dock at the lateral lumen (conspicuous in varicosities), thereby shaping the lumen's cylindrical tunnel structure and transmitting fluid pressure between varicosities that maintains lumen diameter and supports its anterior-posterior extension. .

SK Lange et al. (1999) International C. elegans Meeting "Identification of a gene required for pharyngeal elongation and deleted by mnDf90."

SK Lange, SE Mango

[International C. elegans Meeting, 1999]

Midway during embryogenesis the pharyngeal precursors assemble into a ball of cells that comprises the pharyngeal primordium. Over the next 1-2 hours the primordium elongates to form a tube that is connected to the buccal cavity. We have analyzed the phenotypes of homozygous deficiencies to identify loci involved in this process (see abstract by Portereiko, Domeier and Mango). We found that mnDf90 embryos fail to undergo the initial stages of pharyngeal elongation and never attach to the buccal cavity. Other aspects of pharynx development appear normal: the correct number of pharyngeal cells are present, the pharynx appears to differentiate normally, and MH27 staining indicates that the pharyngeal muscles are properly organized. To isolate the gene(s) responsible for the mnDf90 pharynx defect we screened 3100 haploid genomes for lethal mutations that map to the mnDf90 region. We identified three alleles: px3, px4 and px5 . The most severe allele is px4 , which arrests as a 2-fold embryo with an unelongated but differentiated pharynx. Homozygous px3 embryos arrest at the 3-fold or L1 stages. The pharyngeal phenotype of px3 resembles that of px4 . Embryos mutant for the third allele, px5 , have increased numbers of cell corpses in addition to the unelongated pharyngeal phenotype. Our immediate goals are to determine whether these alleles define one or two different genes and to isolate additional alleles.

Vida Praitis et al. (2005) International Worm Meeting "A temperature-sensitive mutation that affects pharyngeal morphogenesis"

Angela Schacht, Zelealem Yilma, Lensa Yohannes, Michael Miller, Juliette Mushi, Vida Praitis, Sarah Kniss

[International Worm Meeting, 2005]

In C. elegans, pharyngeal morphogenesis is divided into several discreet steps including the creation of a pharyngeal primordium, reorientation of anterior pharyngeal cells, formation of stable epithelium between the buccal cells, pharynx, and intestine, and changes in cell shape (Portereiko & Mango, 2001). We have identified a temperature-sensitive embryonic lethal mutation which causes defects in pharyngeal morphogenesis, resulting in embryos with the pun (pharynx unattached) phenotype. Genetic experiments show the ru5 mutation is weakly semi-dominant and the phenotype can be maternally or zygotically rescued. ru5 embryos grown at the restrictive temperature undergo normal embryonic development until the bean stage when pharyngeal morphogenesis fails to occur properly. Temperature shift experiments show the gene product is required from 4 to 6 hours after the two-cell stage, during morphogenesis, but is otherwise not required for viability. To further characterize the ru5 phenotype, we examined the expression pattern of proteins found in the pharynx and head hypodermis during this stage of development. Our results indicate that early developmental events and the establishment of the pharyngeal primordium occur normally in ru5 embryos grown at the restrictive temperature. Reorientation of the anterior pharynx also appears normal, as the expression patterns of anterior markers such as AJM-1 and SMA-1 in ru5 embryos are indistinguishable from wild type. However, the localization of these proteins in the arcade cells and in the head hypodermis are abnormal and the invagination associated with the buccal cavity does not occur in ru5 embryos. These results suggest the failure to form a stable epithelium is due to defects in the organization, movement, or shape of the arcade cells or anterior hypodermis. Using conventional and snp-snp mapping strategies, we mapped the ru5 mutation to a small interval on LG I. We are currently working on cosmid rescue experiments to identify the altered gene product.