Podbilewicz B (1993) International C. elegans Meeting "To fuse or not to fuse: a developmental question."

Podbilewicz B

[International C. elegans Meeting, 1993]

Podbilewicz B (1997) International C. elegans Meeting "GENETIC ANALYSES OF CELL FUSION"

Podbilewicz B

[International C. elegans Meeting, 1997]

We all know that there are 959 somatic nuclei in the adult hermaphrodite. It has been shown that only 658 cells are mononucleated. Thus, 301 somatic cells undergo programmed cell fusion to form 46 multinucleated cells during specific times of development in C. elegans. To understand the functions of cell fusion we characterized the stereotyped sequence of cell fusions in the wild-type hypodermis and based on this we developed a novel immunofluorescence screen to identify mutants in cell fusion. We obtained embryonic lethal mutations with irregular hypodermis that look like jigsaw puzzles. This class of mutations probably identifies genes required for the specificity of cell fusions. Other classes of embryonic lethal mutations resulted in embryos with more fusions than in wild-type, disorganized hypodermis and defects in elongation. These groups of mutations could probably cause defects in the timing in which cells fuse. We perform phenotypic analyses to determine what aspects of cell fusion during epithelia development are affected by different mutations and the specificity of the resultant defects.

Podbilewicz B et al. (1998) European Worm Meeting "Vulval morphogenesis in C. elegans"

Kishore RS, Podbilewicz B

[European Worm Meeting, 1998]

Cell-cell recognition, fusion, migration and rearrangements are processes involved in organ formation and patterning. In C. elegans these have been reported to occur in the formation of the hypodermis, uterus, excretory gland cells, pharynx and vulva. Cell migrations and fusions during vulval development were characterized using immunofluorescence techniques and confocal microscopic reconstructions. Vulval sections at different times during the developmental process were stained for MH27, an adherens junction protein (1). Events have been followed from the time the fates of the six vulval precursor cells (VPCs) have been specified through inductive signalling by the anchor cell to the construction of a tube that consists of seven toroidal rings stacked together (a total of 20 hr.). These rings are designated as: vulA, vulB1, vulB2, vulC, vulD, vulE and vulF (2,3). Early morphogenesis involves generation of the VPC daughters, VPC grand-daughters and great-granddaughters, which results in 22 epithelial cells forming a vulva primordium. These cells are more or less arranged in a two dimensional fashion. Later, these cells perform short range migrations from the outer regions towards the central midline of the developing vulva in a symmetrical fashion. They also undergo fusions with specific partners before they migrate, eg. precursors of vulA on either side of the primordium fuse in pairs before migrating. The cells adjacent to the innermost cells form processes and migrate around them. This is repeated in a temporal sequence by the next outer cells which send processes around their inner neighbours, which meet at the midline. These migrations push the inner ring precursors dorsally, forming an invagination. The final result is the formation of a stack of seven rings (vulA to vulF). This is followed by fusion between the component cells in each ring, the order of the intra-toroidal fusions being vulD,vulA, vulC, vulF and vulE. The anchor cell eventually penetrates the inner ring (vulF) and fuses with the uterine cell, utse (4). In the late L4 stage, the vulva is connected to the uterus by vulF and vulval muscles attach the vulva to the body wall and the uterus. In summary, vulval development involves specific migrations and fusions of the cells involved in a spatial and temporal order. Our preliminary studies on the Muv mutant lin-15(e1763) which has one !real! vulva and 2-3 !pseudovulvae! have shown early L4 worms where the vulval ring precursor cells have failed to migrate, and form ring-like processes around their inner neighbours thus failing to produce the stack of rings in the center. Further studies with this mutant and other Muv mutants will explain the mutant phenotype in terms of specific defects in migration and fusions of the cells involved. 1. Podbilewicz, B. and White, J.G. (1994). Dev.Biol. 161, 408-424. 2. White, J.G. Personal Communication. 3. Sharma-Kishore, R. and Podbilewicz, B. (1997).The Worm Breeders Gazette. Vol.15#1. p58. 4. Newman, A.P., White, J.G. and Sternberg, P.W. (1996). Development 122, 3617-3626.

Podbilewicz B (1996) European C.elegans Meeting "Cell fusions in pharyngeal muscles, hypodermis, sperm, and ADAMs."

Podbilewicz B

[European C.elegans Meeting, 1996]

Podbilewicz B (1995) International C. elegans Meeting "ADM-1 MEMBRANE PROTEIN CONTAINS SNAKE VENOM METALLOPROTEASE- AND DISINTEGRIN-LIKE DOMAINS AND MAY FUNCTION DURING MEMBRANE FUSIONS IN SHEATH CELLS OF SENSORY ORGANS AND HYPODERMIS"

Podbilewicz B

[International C. elegans Meeting, 1995]

I am interested in understanding the role of cell fusion in development. A glycoprotein located on the surface of guinea pig sperm (PH-30/fertilin) is implicated in sperm-egg fusion and is analogous to viral fusion proteins (Blobel, et al. 1992. Nature. 356:248-252). I will describe the isolation, molecular and cellular characterization of the first reported invertebrate gene related to PH-30 in C. elegans (adm-1, for A Disintegrin and a Metalloprotease domain). ADM-1 is related to a family of mammalian proteins containing snake venom disintegrin- and metalloprotease-like domains. ADM-1 contains six domains: prepro, metalloprotease, disintegrin, cysteine-rich with putative fusion peptide and epidermal growth factor-like repeat, transmembrane, and cytoplasmic. In immunoblots two major forms of ADM-1 are recognized, these proteins may represent precursor and mature forms of the protein (115 and 30 kD respectively) or could be the result of alternative splicing. Using immunofluorescence and in situ hybridization the products of adm-1 have been detected in the hypodermis and in the sheath cells of cuticular sensory organs. The hypodermis is a syncytial tissue formed by cell fusions during development. The sheath cells are active in intracellular transport suggesting that adm-1 could have a role in fusions during intracellular organelle biogenesis and in cell-to-cell fusions in the hypodermis. I will discuss how ADM-1 could be involved in intra- or intercellular fusions, and/or in the development and maintenance of the sheath cells in sensilla and the hypodermis.

B Podbilewicz et al. (1999) International C. elegans Meeting "Cold sensitive fusion: kinetic dissection of cell fusion during morphogenesis in N2 and duf-1"

T Gattegno, B Podbilewicz, Y Rabin, M Glickman

[International C. elegans Meeting, 1999]

Cell fusion is an essential process in development conserved throughout evolution. We have analysed the sequence of epithelial cell fusions in C. elegans [1] and have isolated mutations that disrupt cell fusion in the embryo. To elucidate the molecular mechanisms of cell fusion in situ we arrested or slowed down this process by lowering the temperature. We used confocal microscopy and a custom-made cooling stage to record 4D-timelapse movies of developing embryos expressing MH27-GFP fusion protein in the adherens junctions[2] at different temperatures. At temperatures below 4 o C, comma stage embryos did not elongate and epithelial fusions did not occur. However, at 5 o C embryos elongated to 2-fold at a rate of about 20-fold slower than that at 20 o C and dorsal cell fusions did not occur. Moreover, this treatment phenocopies the arrest of duf-1(zu316cs) embryos at 15 o C[3]. The fusion rate measured from timelapse recordings as disappearance of MH27-GFP staining varied from 50 nm/min at 6 o C to 1000 nm/min at 27.5 o C. Thus, we show that the cell fusion rate is strongly dependent on temperature. Using a combination of kinetic and genetic analyses we have initiated the dissection of the multi-step process involving the opening and expansion of fusion pores necessary for morphogenesis and the formation of organs in C. elegans and other organisms. We propose that cell fusion in C. elegans can be dissected into at least three steps: (1) transient hemifusion; (2) rearrangements of two lipid bilayers into one (fusion pore formation arrested at 5 o C); and (3) expansion of fusion pore (estimated fusion rates at 6 o C). Using Arrhenius plots for N2 and duf-1 we can get information on the mechanisms of pore expansion during cell fusion in vivo. The energy of activation can be estimated and compared between wild-type and mutant. [1] Podbilewicz B. and White J.G. (1994) Dev. Biol. 161, 408-424. [2] Mohler, W.A et al (1998) Curr. Biol. 8, 1087-1090. [3] Gattegno T. (1999) IWM Abstract.

Gattegno T et al. (1998) European Worm Meeting "duf-1(zu316) a mutation affecting cell fusion"

Podbilewicz B, Gattegno T

[European Worm Meeting, 1998]

During the development of Caenorhabditis elegans, about 30% of the somatic cells fuse to form multi-nucleated cells. Embryonic fusion events occur at the epithelial monolayer that surrounds the embryo. The fusion pattern in the wild type is being used as a basis to compare fusion event patterns in mutants that are affected in the fusion process. Characterization of duf-1 (zu316) dorsal unfused, C.elegans first hypofused mutant provides an opportunity to clone genes that are involved in the process or in its regulation. duf-1(zu316) was proven to be a recesive lethal mutation. Embryos homozygous to the mutation stop developing during elongation (1.5 fold to 2.0 fold) and die. The mutation was mapped to the left arm of chromosome X. The fusion defect was defined by comparing the fusion pattern of the duf-1 mutant to the wild type pattern and it was characterized by immunofluorescence using MH27 antibody (a monoclonal antibody that stains epithelial cell borders). When fusions occur between epithelial cells, there is a rearrangement of the staining pattern of the antibody, providing a way to observe fusion events. One of the main questions concerns the specificity of the fusion defect. Embryos homozygous to the duf-1 (zu316) mutation were stained using monoclonal antibodies that specifically stained five different tissues (intestine, pharynx, body wall muscle, seam cells and epithelial cells). The staining patterns were compared to the wild type patterns for embryos at similar stages. The defect in embryos homozygous to duf-1(zu316) is specific to dorsal hypodermal fusion events. The mutant was found to be cold sensitive, when grown at 15!C, duf-1 embryos arrest at 1.5 to 2.0 fold stages of elongation, with a twist at the tail and fewer fusions at the dorsal epithelium. When grown at 25!C, duf-1 embryos arrest at 3.0 fold stages of elongation, some hatch, fusion events occur normally and a twist at the posterior part was observed. The cold sensitivity implies that duf-1 (zu316) is not the null allele. duf-1(zu316) was crossed to a trangenic strain that contains the fusion sequence MH27gene::GFP (Green Fluorescence Protein) integrated to its genome. The strain duf-1(zu316); MH27::GFP was isolated, it enables the characterization of fusion events in live embryos, with no need to do immunofluorescence. We plan to mutagenize this strain and screen for mutants that interact with duf-1 geneticaly. The research results are another step towards the identification of the molecular events that lead to regulated fusion events in C.elegans. 1 Podbilewicz B. (1997) Worm Meeting Abstract, p.300. 2 Gattegno T. and Podbilewicz B. (1997) Worm Meeting Abstract, p.546. 3 Podbilewicz B. and White, J.G (1994). Dev.Biol. 161, 408-424. 4 Kindly provided by J.Simske and J.Harding.

Gattegno T et al. (1997) International C. elegans Meeting "A MUTATION AFFECTING CELL FUSIONS DURING ELONGATION OF THE EMBRYO"

Gattegno T, Podbilewicz B

[International C. elegans Meeting, 1997]

Cell fusion is a widespread process throughout development and evolution. Many organs in multicellular organisms contain multinucleate cells formed by cell fusion; e.g., in humans (placenta, bone and muscle) and in C. elegans (hypodermis, vulva, pharynx, uterus, and excretory gland). Enveloped viruses (e.g. HIV) fuse with cell membranes in order to invade target cells, and fertilization involves sperm-egg fusion. We have characterized the order of epithelial cell fusions in C. elegans (1) and have isolated mutations that disrupt cell fusions in the embryo. In our pilot screens we did not obtain any mutation with fewer fusions than in wild-type. The zygotic lethal mutation zu316 was isolated in a screen for mutations that arrest during embryonic elongation (2). In the strain JJ825 zu316/+; lin-2 (e1309) the dorsal hypodermal cells fail to fuse. This mutant was outcrossed and we are now characterizing zu316 by 4D microscopy and immunofluorescence using tissue-specific mAbs and confocal microscopy. The development of the mutant terminates between 1.5 to 2-fold stages of elongation but some embryos arrest earlier. The mutants appear normal up to the beginning of morphogenesis and then the dorsal hypodermal cells look round during elongation and do not get flattened as in N2. Using MH27 and anti-LIN26 antibodies (kindly provided by R. Waterston and M. Labouesse, respectively) we notice that in zu316 there are fewer cell fusions in the dorsal hyp6 and hyp7 than in wild-type embryos. 1. Podbilewicz B. and White J.G. (1994) Dev. Biol. 161, 408-424 2. Costa M. and Priess J. (1995) Worm Meeting Abstract, p. 165 We thank Mike Costa and Jim Priess for sending JJ825 to us.

T Gattegno et al. (1999) International C. elegans Meeting "duf-1(zu316cs) - Dorsal UnFused"

B Podbilewicz, T Gattegno

[International C. elegans Meeting, 1999]

During embryonic development of C.elegans , hypodermal cells undergo programmed cell fusion to create multi-nucleated cells. Though the temporal and spatial patterns of the fusion events in the wild type have been characterized the molecular mechanism that induces cell fusion is unclear(1). Analysis of mutants affected in the fusion process provides an opportunity to clone the genes involved. duf-1(zu316cs) has fewer fusion events in the dorsal hypodermis compared to wild type(2) and it was isolated during a screen for elongation defects(3). Embryonic elongation occurs simultaneously with a series of fusion events at the hypodermis. We have characterized zu316cs as a zygotic lethal cold sensitive mutation and mapped it to the left arm of chromosome X, between -15.81 and -17.26 in the genetic map. The arrest phenotype was characterized using Nomarski optics; arrested embryos continue to move and have a twisted tail(2). Fusion analysis was done using the monoclonal antibody MH27 that recognizes the apical boundaries of hypodermal cells(1); a strain expressing MH27-GFP fusion protein(4) and carrying zu316cs enables efficient characterization of living embryos. MAbs indicative for muscles, intestine, pharynx, glial-like cells and hypodermis were used to characterize zu316cs . Staining patterns were fundamentally normal implying that zu316cs is specifically affected in the hypodermal fusion process. We further analyzed the number and identity of the unfused cells by counting junctions in arrested embryos (7.6+/-3.2; n=61) in comparison to wild type embryos at similar elongation stages (1.5+/-0.5; n=15). zu316cs arrest phenotype at the restrictive temperature can be artificially created by growing wild type embryos at 5degC(5). These results suggest conditional relationship between elongation and fusion; elongation does not proceed beyond 2 fold when hypodermal fusion is affected. Phenotypic and molecular characterization of duf-1 will contribute to a better understanding of the association between elongation and fusion processes. [1] Podbilewicz B. and White J.G. (1994) Dev. Biol. 161, 408-424. [2] Gattegno T. and Podbilewicz B. (1997) IWM Abstract, p.546. [3] Costa M. and Priess J. (1995) IWM Abstract, p.165. [4] Mohler W.A., et al. (1998) Curr Biol. 8, 1087-1090. [5] Podbilewicz B. (1999) IWM Abstract.

Adir V et al. (1997) International C. elegans Meeting "ADM-2 A NEW MEMBER OF THE ADAM FAMILY WITH AN ACTIVE METALLOPROTEASE DOMAIN"

Podbilewicz B, Adir V

[International C. elegans Meeting, 1997]

The ADAMs gene family encodes transmembrane proteins which contain both disintegrin and metalloprotease domains. Some ADAMs have a metalloprotease like domain but lack the active site, while others have a consensus active site sequence for a zinc dependent metalloprotease as in snake venoms. The ADAMs gene family includes fertilin a and b (involved in sperm-egg fusion), meltrin-a (required for myotube formation in mice), TACE (a TNF-a converting enzyme), and KUZ (the kuzbanian gene product essential for neural fate in Drosophila). Other members of this family may be involved in different cell-cell and cell-matrix interactions. ADM-1, was the first gene from this family found in C. elegans, and is expressed in cells that undergo cell fusion. However, ADM-1 lacks the consensus active site sequence for a zinc dependent metalloprotease. In order to identify new genes related to the ADAMs gene family in C. elegans, we used the TBLASTN algorithm and found the cDNA clone yk35e9 which encodes adm-2. Alignment of ADM-2 with the metalloprotease domains of other ADAMs, shows that the consensus active site is highly conserved. adm-2 was mapped to YACs Y50F10 and Y49B10 and to the cosmid C04A11. Consensus HEXXHXXGXXH- ADM-2 HELGHDFGNDHD fertilin a HELGHNLGIRHD (Blobel et al., Nature, 1992) meltrin a HELGHNFGMNHD (Yagami Hiromasa et al., Nature, 1995) TACE HELGHNFGAEHD (Black et al., Nature, 1997) KUZ HEIGHNFGSPHD (Rooke et al., Science, 1996) adamalysin HELGHNLGMEHD (Gomis-Ruth et al., EMBO J., 1993) ADM-1 QSIGHLLGLEHD (Podbilewicz, Mol. Biol. Cell, 1996) Alignment of the consensus active sites for zinc-dependent metalloproteases and ADM-1. To study the function of adm-2 and its effect on C. elegans development we inject adm-2 antisense RNA into wild type nematodes. We also perform rescue experiments to candidate mutants that mapped genetically to the genome location of adm-2.