Biology

Cucumbita leaf, Nerium leaf and Water Hyacinth samples are gathered from the vegetative ground of the School's lagoon, The Bougainvillea twigs, Coleus blumei and Coleus aromaticus are gathered from Provident Village, Marikina City and the Allium cepa and Zea mays sample are collected through germination at home. All the samples are washed by tap water prior to the specimen preparation.

Cell-cell fusion is inherent to sexual reproduction. Loss of HAPLESS 2/GENERATIVE CELL SPECIFIC 1 (HAP2/GCS1) proteins results in gamete fusion failure in diverse organisms, but their exact role is unclear. In this study, we show that Arabidopsis thaliana HAP2/GCS1 is sufficient to promote mammalian cell-cell fusion. Hemifusion and complete fusion depend on HAP2/GCS1 presence in both fusing cells. Furthermore, expression of HAP2 on the surface of pseudotyped vesicular stomatitis virus results in homotypic virus-cell fusion. We demonstrate that the Caenorhabditis elegans Epithelial Fusion Failure 1 (EFF-1) somatic cell fusogen can replace HAP2/GCS1 in one of the fusing membranes, indicating that HAP2/GCS1 and EFF-1 share a similar fusion mechanism. Structural modeling of the HAP2/GCS1 protein family predicts that they are homologous to EFF-1 and viral class II fusion proteins (e.g., Zika virus). We name this superfamily Fusexins: fusion proteins essential for sexual reproduction and ...

A new experimental apparatus for temperature-controlled microscopy has been developed for the study of the temperature dependency of developmental processes in the nematode Caenorhabditis elegans. However, the application of this apparatus is rather general and can be used for a wide range of temperatures between 2 10 and 90 8C. The new apparatus is easy to use, inexpensive, simple to construct, and is designed for precise temperature control of oilimmersion microscopy using epifluorescence. Thermal analysis of the experimental apparatus shows the effects of each of its components, as well as the effects of uncertainty in temperature measurements. Finally, results of this study indicate that: (i) embryos incubated and imaged at temperatures of 8 8C and below do not elongate; (ii) the initial elongation rate is strongly temperature-dependent between 9 and 25 8C.

A search was carried out for homologues of possible fusogenic proteins to study their function in a genetically tractable animal. The isolation, molecular, and cellular characterization of the Caenorhabditis elegans adm-1 gene (a disintegrin and metalloprotease domain) are described. A glycoprotein analogous to viral fusion proteins has been identified on the surface of guinea pig sperm (PH-30/fertilin) and is implicated in sperm-egg fusion. adm-1 is the first reported invertebrate gene related to PH-30 and a family of proteins containing snake venom disintegrin-and metalloprotease-like domains. ADM-1 shows a domain organization identical to PH-30. It contains prepro, metalloprotease, disintegrin, cysteine rich with putative fusion peptide, epidermal growth factor-like repeat, transmembrane, and cytoplasmic domains. Antibodies which recognize ADM-1 protein in immunoblots were generated. Using immunofluorescence and in situ hybridization, the products of adm-1 have been detected in specific cells during different stages of development. The localization of ADM-1 to the plasma membrane of embryonic cells and to the sheath cells of sensory organs suggests a function in cell adhesion. ADM-1 expression in the hypodermis, pharynx, vulva, and mature sperm is consistent with a putative role in somatic and gamete cell fusions.

Injury triggers regeneration of axons and dendrites. Research identified factors required for axonal regeneration outside the CNS, but little is known about regeneration triggered by dendrotomy. Here we study neuronal plasticity triggered by dendrotomy and determine the fate of complex PVD arbors following laser surgery of dendrites. We find that severed primary dendrites grow towards each other and reconnect via branch fusion. Simultaneously, terminal branches lose self-avoidance and grow towards each other, meeting and fusing at the tips via an AFF-1-mediated process. Ectopic branch growth is identified as a step in the regeneration process required for bypassing the lesion site. Failure of reconnection to the severed dendrites results in degeneration of the distal end of the neuron. We discover pruning of excess branches via EFF-1 that acts to recover the original wild-type arborization pattern in a cell-autonomous process. In contrast, AFF-1 activity during dendritic auto-fusion...

Mammalian sperm–egg adhesion depends on the trans-interaction between the sperm-specific type I glycoprotein IZUMO1 and its oocyte-specific GPI-anchored receptor JUNO. However, the mechanisms and proteins (fusogens) that mediate the following step of gamete fusion remain unknown. Using live imaging and content mixing assays in a heterologous system and structure-guided mutagenesis, we unveil an unexpected function for IZUMO1 in cell-to-cell fusion. We show that IZUMO1 alone is sufficient to induce fusion, and that this ability is retained in a mutant unable to bind JUNO. On the other hand, a triple mutation in exposed aromatic residues prevents this fusogenic activity without impairing JUNO interaction. Our findings suggest a second function for IZUMO1 as a unilateral mouse gamete fusogen.

Background: A surprising amount of developmental variation has been observed for otherwise highly conserved features, a phenomenon known as developmental system drift. Either stochastic processes (e.g., drift and absence of selection-independent constraints) or deterministic processes (e.g., selection or constraints) could be the predominate mechanism for the evolution of such variation. We tested whether evolutionary patterns of change were unbiased or biased, as predicted by the stochastic or deterministic hypotheses, respectively. As a model, we used the nematode vulva, a highly conserved, essential organ, the development of which has been intensively studied in the model systems Caenorhabditis elegans and Pristionchus pacificus. Results: For 51 rhabditid species, we analyzed more than 40 characteristics of vulva development, including cell fates, fate induction, cell competence, division patterns, morphogenesis, and related aspects of gonad development. We then defined individual characters and plotted their evolution on a phylogeny inferred for 65 species from three nuclear gene sequences. This taxon-dense phylogeny provides for the first time a highly resolved picture of rhabditid evolution and allows the reconstruction of the number and directionality of changes in the vulva development characters. We found an astonishing amount of variation and an even larger number of evolutionary changes, suggesting a high degree of homoplasy (convergences and reversals). Surprisingly, only two characters showed unbiased evolution. Evolution of all other characters was biased. Conclusions: We propose that developmental evolution is primarily governed by selection and/or selection-independent constraints, not stochastic processes such as drift in unconstrained phenotypic space.

Background: The molecular mechanisms that determine axonal growth potential are poorly understood. Intrinsic growth potential decreases with age, and thus one strategy to identify molecular pathways controlling intrinsic growth potential is by studying developing young neurons. The programmed and stereotypic remodeling of Drosophila mushroom body (MB) neurons during metamorphosis offers a unique opportunity to uncover such mechanisms. Despite emerging insights into MB g-neuron axon pruning, nothing is known about the ensuing axon re-extension. Results: Using mosaic loss of function, we found that the nuclear receptor UNF (Nr2e3) is cell autonomously required for the re-extension of MB g-axons following pruning, but not for the initial growth or guidance of any MB neuron type. We found that UNF promotes this process of developmental axon regrowth via the TOR pathway as well as a late axon guidance program via an unknown mechanism. We have thus uncovered a novel developmental program of axon regrowth that is cell autonomously regulated by the UNF nuclear receptor and the TOR pathway. Conclusions: Our results suggest that UNF activates neuronal re-extension during development. Taken together, we show that axon growth during developmental remodeling is mechanistically distinct from initial axon outgrowth. Due to the involvement of the TOR pathway in axon regeneration following injury, our results also suggests that developmental regrowth shares common molecular mechanisms with regeneration following injury.

cell fusion events in C. elegans [5, 10, 16-18] and Drosophila [4, 19, 20]. However, little is known regarding the underlying molecular details. Most of what is known about membrane fusion came from studying relatively simple fusion reactions medi

Directed cell rearrangements occur during gastrulation, neurulation, and organ formation. Despite the identification of developmental processes in which invagination is a critical component of pattern formation, little is known regarding the underlying cellular and molecular details. Caenorhabditis elegans vulval epithelial cells undergo morphological changes that generate an invagination through the formation of seven stacked rings. Here, we study the dynamics of ring formation during multivulva morphogenesis of a let-60/ras gain-of-function mutant as a model system to explore the cellular mechanisms that drive invagination. The behavior of individual cells was analyzed in a let-60/ras mutant by threedimensional confocal microscopy. We showed that stereotyped cell fusion events occur within the rings that form functional and nonfunctional vulvae in a let-60/ras mutant. Expression of let-60/ras gain-of-function results in abnormal cell migration, ectopic cell fusion, and structural fate transformation. Within each developing vulva the anterior and posterior halves develop autonomously. Contrary to prevailing hypotheses which proposed three cell fates (1°, 2°, and 3°), we found that each of the seven rings is a product of a discrete structural pathway that is derived from arrays of seven distinct cell fates (A, B, C, D, E, F, and H). We have also shown how autonomous ring formation is the morphogenetic force that drives invagination of the vulva.

Much less is known about the mechanisms that mediand Benjamin Podbilewicz 2,4 ate plasma membrane fusion in fertilization and syncy-1 Department of Genetics tium formation (Shemer and Podbilewicz, 2000). Fertilins and Developmental Biology and meltrins, members of the ADAM family of membrane University of Connecticut Health Center glycoproteins, are no longer considered candidate fuso-Farmington, Connecticut 06030 gens (Cho et al., 2000; Hernandez et al., 1996). Syncytin, 2 Department of Biology a proviral gene product expressed in primate placenta, Technion has been shown to act as a fusogen in vitro, but its Haifa 32000 recent appearance in evolution cannot account for the Israel ancient role of cell fusion in placentation (Mi et al., 2000). 3 Laboratory of Molecular Biology Several genes identified in yeast, flies, and mammals University of Wisconsin appear to mediate events that precede membrane fu-Madison, Wisconsin 53706 sion, including cell attachment, cell polarization, and cell wall reorganization (Shemer and Podbilewicz, 2000; Taylor, 2000; Wassarman, 1999). A membrane protein Summary (Prm1p) may facilitate actual lipid bilayer fusion in yeast, although it is not absolutely required (Heiman and Wal-Multinucleate cells are widespread in nature, yet the ter, 2000). mechanism by which cells fuse their plasma mem-To identify molecules acting in developmental cell fubranes is poorly understood. To identify animal fusosion, we used Caenorhabditis elegans, a genetic animal gens, we performed new screens for mutations that system where cell fusion occurs in a variety of tissues abolish cell fusion within tissues of C. elegans and the process can be easily observed. One third of throughout development. We identified the gene eff-1, somatic cells fuse in a fully characterized and reproducwhich is expressed as cells acquire fusion compeible pattern during C. elegans development (Podbiletence and encodes a novel integral membrane protein. wicz, 2000; Podbilewicz and White, 1994; Sulston and EFF-1 sequence motifs suggest physicochemical ac-Horvitz, 1977; Sulston et al., 1983). Most fusions are tions that could cause adjacent bilayers to fuse. Mutabetween cells that constitute polarized epithelia (Figure tions in the extracellular domain of EFF-1 completely 1). Several transcription factors are known to regulate block epithelial cell membrane fusion without affectspecific cell fusion events in C. elegans (Alper and Kening other prefusion events such as cell generation, yon, 2001; Ch'ng and Kenyon, 1999; Clark et al., 1993; patterning, differentiation, and adhesion. Thus, EFF-1 Koh and Rothman, 2001). However, no proteins involved is a key component in the mechanism of cell fusion, in the actual membrane fusion event have been identia process essential to normal animal development. fied. In this paper, we isolate two mutations that specifically block cell fusion in all epidermal and vulval epithelia

During cell-cell fusion, two cells' plasma membranes merge, allowing the cytoplasms to mix and form a syncytium. Little is known about the mechanisms of cell fusion. Here, we asked whether eff-1, shown previously to be essential for fusion in Caenorhabditis elegans, acts directly in the fusion machinery. We show that expression of EFF-1 transmembrane protein drives fusion of heterologous cells into multinucleate syncytia. We obtained evidence that EFF-1-mediated fusion involves a hemifusion intermediate characterized by membrane mixing without cytoplasm mixing. Furthermore, syncytiogenesis requires EFF-1 in both fusing cells. To test whether this mechanism also applies in vivo, we conducted genetic mosaic analysis of C. elegans and found that homotypic epidermal fusion requires EFF-1 in both cells. Thus, although EFF-1-mediated fusion shares characteristics with viral and intracellular fusion, including an apparent hemifusion step, it differs from these reactions in the homotypic organization of the fusion machinery.

Developmental cell fusion is found in germlines, muscles, bones, placentae, and stem cells. In Caenorhabditis elegans 300 somatic cells fuse during development. Although there is extensive information on the early intermediates of viral-induced and intracellular membrane fusion, little is known about late stages in membrane fusion. To dissect the pathway of cell fusion in C. elegans embryos, we use genetic and kinetic analyses using live-confocal and electron microscopy. We simultaneously monitor the rates of multiple cell fusions in developing embryos and find kinetically distinct stages of initiation and completion of membrane fusion in the epidermis. The stages of cell fusion are differentially blocked or retarded in eff-1 and idf-1 mutants. We generate kinetic cell fusion maps for embryos grown at different temperatures. Different sides of the same cell differ in their fusogenicity: the left and right membrane domains are fusion-incompetent, whereas the anterior and posterior me...

Making Menorahs Highly branched neuronal processes are typical of numerous neurons. The mechanisms controlling the formation, maintenance and regeneration of neuronal trees are poorly understood. Oren-Suissa et al. (p. 1285 , published online 6 May) observed the development of extensively arborized neurite trees—menorahs—in two highly branched mechanosensory neurons in Caenorhabditis elegans . EFF-1, a fusion protein known to be important for cell fusion, played a key role in arborization.

Background: Maximum Intensity Projections (MIP) of neuronal dendritic trees obtained from confocal microscopy are frequently used to study the relationship between tree morphology and mechanosensory function in the model organism C. elegans. Extracting dendritic trees from noisy images remains however a strenuous process that has traditionally relied on manual approaches. Here, we focus on automated and reliable 2D segmentations of dendritic trees following a statistical learning framework. Methods: Our dendritic tree extraction (DTE) method uses small amounts of labelled training data on MIPs to learn noise models of texture-based features from the responses of tree structures and image background. Our strategy lies in evaluating statistical models of noise that account for both the variability generated from the imaging process and from the aggregation of information in the MIP images. These noisy models are then used within a probabilistic, or Bayesian framework to provide a coarse 2D dendritic tree segmentation. Finally, some post-processing is applied to refine the segmentations and provide skeletonized trees using a morphological thinning process. Results: Following a Leave-One-Out Cross Validation (LOOCV) method for an MIP databse with available "ground truth" images, we demonstrate that our approach provides significant improvements in tree-structure segmentations over traditional intensity-based methods. Improvements for MIPs under various imaging conditions are both qualitative and quantitative, as measured from Receiver Operator Characteristic (ROC) curves and the yield and error rates in the final segmentations. In a final step, we demonstrate our DTE approach on previously unseen MIP samples including the extraction of skeletonized structures, and compare our method to a state-of-the art dendritic tree tracing software. Conclusions: Overall, our DTE method allows for robust dendritic tree segmentations in noisy MIPs, outperforming traditional intensity-based methods. Such approach provides a useable segmentation framework, ultimately delivering a speed-up for dendritic tree identification on the user end and a reliable first step towards further morphological characterizations of tree arborization.