Gattegno, Tamar et al. (2013) International Worm Meeting "Menorah-menorah auto-fusion as a mechanism of PVD response to injury."

Gattegno, Tamar, Podbilewicz, Benjamin, Oren, Meital

[International Worm Meeting, 2013]

The PVDs are two nociceptors localized along C. elegans lateral sides, enabling them to sense strong mechanical stimuli such as pain, temperature and posture. The PVDs develop through a dynamic process which involves extension and retraction of dendrite branches. Though we now better understand C. elegans PVD development, very little is known about PVD regeneration and degeneration following injury. In order to study the genetic pathway guiding PVD response to injury, we use laser microsurgery to severe the PVD and live imaging of GFP reporters. We found that when the PVDs are injured at the 1ry branch, AFF-1 mediated fusion occurs between the terminal branches to bypass the site of injury, followed by EFF-1 mediated pruning and arbor simplification. Degeneration of the distal stump occurred in animals that failed to undergo reconnection of the 1ry severed branch or menorah-menorah fusion. We used Kaede photo-convertible fluorescent protein expressed in the PVD to support the observation that injured PVD has reconnected through auto-fusion. We severed the PVD 1ry branch, recovered the worms and detected reconnection after 6-12 hours. The PVD cell body of recovered animals was than photo-converted from green to red and we analyzed the transfer of the Red-Kaede through the reconnected neuron. We found that PVD reconnection occurs through fusion. We then performed a similar experiment in lin-22 animals which have 2 to 5 extra PVD cell bodies (kindly provided by M. Heiman and C. Yip) to study whether adjacent PVDs connect by fusion. We found that following injury different PVDs can fuse as a response to injury. These experiments show that PVD neurons response to dendrotomy involves four steps: (1) Loss of self-avoidance or tiling. (2) Menorah-menorah fusion mediated by AFF-1 and/or reconnection of injured 1ry branches by auto-fusion. (3) Ectopic sprouting of dendrites from the PVD. (4) Simplification by branch retraction mediated by EFF-1. The outcome of the regeneration process is the recovery of the PVD morphology and if regeneration by auto-fusion fails the distal part degenerates.

Gattegno, Tamar et al. (2015) International Worm Meeting "AFF-1 fuses terminal branches non cell-autonomously during dendritic regeneration."

Oren-Suissa, Meital, Podbilewicz, Benjamin, Kravtsov, Veronika, Gattegno, Tamar

[International Worm Meeting, 2015]

We wish to reveal the basic requirement for dendritic regeneration after injury in order to better understand neuronal plasticity and maintenance. Our model is the PVDs, a pair of highly arborized mechanosensory neurons that are responsible for sensation of strong mechanical stimuli, temperature and posture. PVDs develop through a dynamic process forming its structural units termed "menorahs" that define the resolution of its receptive field (Oren-Suissa et al., 2010). Two cell-cell fusion proteins (EFF-1 and AFF-1) have been identified in C. elegans and they act to mediate different cell fusion events in the hypodermis, pharynx, vulva, uterus, and other organs. EFF-1 has a function to regenerate broken axons by auto-fusion (Ghosh-Roy et al., 2010) and to sculpt PVD dendrites by branch fusion and retraction during development (Oren-Suissa et al., 2010). AFF-1 mediates fusion events of cells whose fusion does not require EFF-1 e.g. anchor cell to utse syncytium and seam cells fusions (Sapir et al., 2007).To understand how dendrites regenerate following injury, we used a femtosecond laser to sever PVD dendrites and followed their regeneration in wild-type and mutant animals. We demonstrate that the reconnection of broken branches occurs through fusion using Kaede photoactivation. PVD dendritic regeneration process involves branch sprouting along with loss of self-avoidance of the menorahs thus allowing fusion of terminal branches to bypass the site of injury. As opposed to wild type, where most of PVD regeneration occurred through terminal branches fusion, in aff-1 mutants PVD regeneration occurred mainly through primary branch fusion. Our results suggest that AFF-1 fuses terminal branches to enable efficient regeneration.We tested whether AFF-1 acts cell autonomously to fuse broken dendrites using tissue-specific rescue experiments. Although AFF-1 expresses in sheath cells of chemosensory neurons in the head we did not observe its expression in the PVD, before or after injury. Moreover, following PVD regeneration of transgenic aff-1 null mutants that express AFF-1 only in the PVD compared to their siblings, we confirmed that AFF-1 can fuse terminal branches; however AFF-1 expression in the PVD could not elevate the number of fused menorahs to wild type level, thus implying that AFF-1 acts non cell-autonomously to reconnect terminal dendrites. This is in contrast to EFF-1, that is expressed in the PVD and acts cell-autonomously to sculpt PVD dendrites. We are currently analyzing PVD regeneration in aff-1 mutants that express AFF -1 in the seam cells and in the hypodermis.

Tamar Gattegno et al. (2003) International Worm Meeting "eff-1 is not alone: Search for new cell-fusion genes in C. elegans"

Nirit Assaf-Reizel, Benjamin Podbilewicz, Tamar Gattegno

[International Worm Meeting, 2003]

Cell fusion is an important developmental process, which occurs in numerous organisms and is abundant in C. elegans epithelia and pharyngeal muscles. The genes that have been identified to be involved in cell fusion are regulatory factors, except for eff-1(epithelial fusion failure), which encodes novel type- I transmembrane proteins, and is a candidate to be involved in the fusion machinery. The zygotic lethal mutation zu316 was isolated in a screen for mutations that arrest during embryonic elongation (Costa M. and Priess J. IWM abstract, 1995). idf-1(irregular dorsal fusion)(zu316) mutation, previously known as duf-1(dorsal unfused), which is specifically affected in dorsal embryonic fusion events, was mapped to the left arm of chromosome X. Two overlapping YACs (Y105G12 and Y59E1) and a common cosmid (T26C11) partially rescued zu316 animals in transformational rescue experiments. However, we have not got idf-1(zu316)-like phenotypes following RNAi targeted to each one of the seven ORFs predicted in the T26C11 cosmid. In order to discover genetic interactions between eff-1 and idf-1 we generated an eff-1(hy21); idf-1(zu316) double mutant and analyzed its phenotypes. The double mutants arrest similar to idf-1 single mutants, whereas the fusion block resembles the pattern in eff-1(hy21) single mutants. This implies that idf-1 may control embryonic dorsal fusion by regulating eff-1 activity and may regulate other processes, which are essential for viability, through other factors (Clari Valansi et al. IWM abstract, 2001). Thus far, three clonal screens designed to find fusion-affected mutants, covering 15000 F1s yielded merely eff-1 alleles (Podbilewicz IWM abstract, 1997, Mohler, W A., Shemer G. et al. 2002). We have made an additional two-step clonal EMS screen of 4500 F1s, resulting in the isolation of two new eff-1 alleles. First, we isolated Pvl, Muv, Dpy and tail-defective animals using a dissecting microscope and second we analyzed these animals for cell-fusion defects, using the fluorescence microscope. hy40 and hy41 are both missense point mutations (Assaf N. IWM abstract, 2003) that display severe Eff phenotypes (Dpy, animals with pommelled tail, protruded vulva and a small brood size). We are currently performing lager scale non-clonal screens looking for new eff-1 alleles and for novel genes involved in cell fusion.

Yuval, Omer et al. (2015) International Worm Meeting "Menorah Analyzer - a MATLAB based software for the analysis of 2D images of the PVD neuron in C.elegans."

Yuval, Omer, Podbilewicz, Benjamin, Shemesh, Tom, Gattegno, Tamar

[International Worm Meeting, 2015]

Nociceptive neurons develop a complex dendritic arbor to sense noxious stimuli, which enables animals to react to environmental insults and perform self-protective behaviors. The arborized mechanoreceptor neuron (PVD) is a multi-dendritic nociceptor neuron in C. elegans. PVDL and PVDR form repeating, non-overlapping dendritic trees, called Menorahs.The morphology of the PVD is thought to be related to its function, with aberrant morphologies associated with impaired physiology. However, no rigorous quantification, protocol or procedure has been developed in order to accurately and uniformly investigate the PVD structures and specifically the menorahs.Here, we present a software tool, Menorah Analyzer, for the automatic analysis of the morphology of the PVD neuron and the menorahs structures. The output of the Analyzer is a quantized reconstruction of the PVD and extraction of key morphological features, such as density of branches, ordering of individual dendrites, as well as length, curvature and alignment of branch segments. Using this automated analysis of the dendritic arbor will enable the generation of a database of various morphologies, as well as to track the time evolution of the structures by processing movies.Ultimately, we will be able to use the software to process any 2D image of the PVD neuron (and perhaps other neurons), and learn about the connection between neuronal morphology and behavior, by comparing quantitative data of WTmutants, youngadult, socialisolated etc.The software accepts as an input one or more 2D images of the PVD neuron - either the original images from the confocal microscope or a manual reconstruction using a tablet.The output, which is displayed in excel tables and graphs, is a reconstruction of each image with different colors representing different branch orders and the extraction of morphological parameters, analyzed per image and as a function of time.Our main biological question is how different neuronal morphologies (junctions, 90o turns etc.) and specifically the Menorahs, form and how are they maintained. We are using the Menorah Analyzer along with developmental genetic techniques in order to answer this question.

Smurova, Ksenia et al. (2009) International Worm Meeting "Phenotypic characterization of cell fusion in eff-1 alleles."

Podbilewicz, Benjamin, Gattegno, Tamar, Assaf-Reizel, Nirit, Smurova, Ksenia

[International Worm Meeting, 2009]

Developmental cell fusion is an important process in eukaryotes. The eff-1 (epithelial fusion failure) gene encodes membrane proteins required for epithelial cell fusion in C. elegans. eff-1 encodes two single-pass integral membrane proteins and two secreted polypeptides by alternative splicing. The transmembrane proteins contain a long extracellular portion, with a TGF-b receptor type 1 domain and 8 pairs of cyteines, a transmembrane domain, and a short intracellular tail. Comparison between the phenotypes caused by different alleles of eff-1 enables us to create an allelic series and deduce the importance of the mutated domains to the function of the proteins. We isolated six viable cell fusion mutants with a broad cell fusion blockage through all developmental stages (hy32, hy40, hy41, hy42, hy43 and hy44). All the new alleles failed to complement eff-1(hy21) and we compared them with eff-1 alleles that were previously characterized (hy21, np29, ok1021 and oj55). Cell junctions in embryos and larvae were visualized using immunofluorescense with MH27 antibodies. We analyzed the projections of 3D stacks that were obtained by confocal microscopy. In all eff-1 mutants, except oj55, hy43 and hy44, all the embryonic hypodermal cells precursors to hyp6 and hyp7 failed to fuse. Surprisingly, we found partial embryonic hypodermal fusion in oj55, hy43 and hy44 embryos. hy40 displayed the most severe Eff-1 phenotype that included Dumpy-Unc animals, deformed tail tip and a complete cell fusion blockage through all stages of development. hy40 resulted in a stop codon at amino acid 224; this short protein represents about half of the extra cellular part of the EFF-1 protein. hy41 gave rise to a missense mutation of the extracellular part of the EFF-1 protein. hy41 also showed rather severe Eff-1 phenotype. hy32 mutation is a cystein 135 change at the TGF-b receptor type 1 like domain of EFF-1. hy32 shows a more severe phenotype than hy21 allele and a complete loss of embryonic and larval cell fusion revealing the importance of the C135 in the structure and function of the fusogenic activity of the extracellular domain and in particular the domain that may fold as a TGF-b receptor type 1 like domain. hy42, hy43 and hy44 showed less severe phenotypes and have variable fusion blockage during development. We are working on the molecular characterization of these mutations.

Clari Valansi et al. (2001) International C. elegans Meeting "GENETIC INTERACTIONS BETWEEN TWO CELL FUSION MUTANTS"

Benjamin Podbilewicz, Tamar Gattegno, Clari Valansi

[International C. elegans Meeting, 2001]

Hypodermal cells undergo programmed cell fusion to generate multi-nucleated cells during embryonic development in C.elegans . Analysis of mutants that have epithelial cells that fail to fuse provides an opportunity to clone the genes involved. duf-1 ( zu316cs ) (Dorsal UnFused) is a zygotic lethal mutant that arrests at 1.5 to 2- fold stage of elongation with fewer dorsal hypodermal cell fusions (1). Strains expressing jam-1 ::GFP fusion protein that is localized to the adherens junctions of epithelial cells were used for fusion analysis on living animals that were also analyzed by Nomarski optics. eff-1 (hy21) is a recessive viable mutant with lumpy, Dpy, Pvl, and Egl phenotype that is defective in all the epithelial cell fusion processes (e.g. vulva, embryonic hypodermis, Pn.p cells, seam cells.) (2) To investigate the possible genetic interactions between eff-1 and duf-1 we constructed and analyzed an eff-1 ( hy21ts ) homozygous duf-1 ( zu316cs ) heterozygous strain. We found that duf-1 /+; eff-1 segregates 25% duf-1;eff-1 embryos that arrest with all hypodermal cells unfused and 75% eff-1 viable unfused animals. Since the duf-1 ; eff-1 arrested embryos had a 100% unfused hypodermis (both dorsal and ventral) and eff-1 ; duf-1 /+ were viable unfused identical to eff-1 , thus, eff-1 is epistatic to duf-1 concerning hypodermal dorsal fusion while duf-1 is also independently required for embryonic viability. In summary, duf-1 embryos arrested with partially unfused dorsal hypodermis while the duf-1; eff-1 embryos arrested with completely unfused hypodermis. Both eff-1; duf-1 /+ and eff-1 worms were viable with the Eff (Epithelial Fusions Failed) phenotype. We propose that duf-1 controls dorsal epidermal fusions by regulating the activity of eff-1 that may be a general Effector of Fusion active in all epithelial cells. 1. Gattegno T. and Podbilewicz B. IWM (1999) Abstract, p. 328. 2. Shemer et al. Mohler et al. (2001) 13 th International C. elegans Meeting. duf-1 ------> eff-1 ------> dorsal hypodermal fusion

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.

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.

Brar, Harjot Kaur et al. (2021) International Worm Meeting "Dendrite regeneration in PVD neuron is controlled by the RAC GTPase CED-10 and the RhoGEF TIAM-1"

Ghosh-Roy, Anindya, Dey, Swagata, Brar, Harjot Kaur, Pandey, Devashish, Bhardwaj, Smriti, Dey, Shirshendu

[International Worm Meeting, 2021]

Neurons are vulnerable to physical insults which compromise the integrity of both dendrites and axons. Unlike well characterized axon regeneration, our knowledge in dendrite regeneration is limited. To understand the mechanisms of dendrite regeneration, we used PVD neurons with stereotyped branched dendrites. Using femtosecond laser, we severed the primary dendrites and axon of this neuron. After the primary dendrite was severed near the cell body, we observed a sprouting of new branches from the cut site within 3 hours. By 24 hours, the primary dendrite regrew to cover the original territory in complex pattern unlike the uninjured dendrites. We quantified the regeneration in broadly two aspects- the territory covered and fusion phenomena (Oren-Suissa et al.,2017, Kravtsov et al.,2017). Axon injury causes a retraction of the severed end followed by a Dual leucine zipper kinase-1(DLK-1) dependent regrowth from the severed end or conversion of neighboring dendrite to axon. However, Dendrite regeneration was independent of DLK-1 and other conventional axon regeneration pathways including cAMP elevation, let-7 miRNA, Akt-1 and Phosphatidyl serine exposure/PS. Among various candidates tested, ced-10 mutants showed a defect in dendrite regeneration. It is a RAC GTPase involved in regulation of neuronal cytoskeleton and cell engulfment. Cell-specific rescue experiments suggest that cell-autonomous and epidermal expression of CED-10 is required for dendrite regrowth and fusion, respectively. Moreover, the PVD-specific expression of an activated version of CED-10 led to both increased branching and fusion. We ventured further to find the upstream players which can control the function of RAC GTPase in dendrite regeneration. We found out that the TIAM-1 RhoGEF is required for dendrite regeneration and the activated CED-10 can bypass the requirement of TIAM-1 in dendrite regrowth. Our work provides a framework for understanding the cellular mechanism of dendrite regeneration using PVD model. Reference: Oren-Suissa, M., T. Gattegno, V. Kravtsov and B. Podbilewicz, 2017 Extrinsic Repair of Injured Dendrites as a Paradigm for Regeneration by Fusion in Caenorhabditis elegans. Genetics 206: 215-230. Kravtsov, V., M. Oren-Suissa and B. Podbilewicz, 2017 The fusogen AFF-1 can rejuvenate the regenerative potential of adult dendritic trees by self-fusion. Development 144: 2364-2374.

Gattegno T et al. (1997) Worm Breeder's Gazette "Characterization of duf-1: C. elegans first Hypofusion Mutant."

Gattegno T, Podbilewicz B

[Worm Breeder's Gazette, 1997]

We are interested in mechanisms of cell adhesion and cell fusion. Cell fusion in humans occurs during fertilization and during the development of multinucleate cells that are contained in bones, muscles and placenta. In C. elegans there are cell fusions during the formation of the vulva, pharynx, uterus, excretory gland and in the hypodermis. There are 46 multinucleate cells (1) of which hyp7 is the largest (133 nuclei).Hpy 7 is part of the hypodermal envelope that surround the adult worm. In a screen for mutations that are affected in the process of cell fusion many mutants were found and analyzed. The mutations were sorted into two main groups: hyperfusion and disordered hypodermis (1). No mutants with fewer fusions in the hypodermis were identified (hypofusion). duf-1(zu316cs) is a dorsal unfused zygotic lethal mutation that was isolated in a screen for mutants affected in the process of elongation (2). The phenotype was analyzed by immunofluoresence using the MH27 antibody. The MH27 protein is expressed in the adherens junctions at the apical domain of the hypodermis and it enable us to follow the fusions between hypodermal cells. After outcrossing we found that zu316cs is a cold sensitive mutation, at 15!C embryonic elongation arrests between 1.5 to 2 fold with fewer fusions in the dorsal hypodermis (hyp6 and hyp7) compared to the wild type and the tail is twisted (3). At 25!C some embryos arrest at 1.5 to 2 fold with fewer dorsal fusions, most embryos arrest at 3 fold and many hatch with normally fused hyp7 and variable posterior defects. duf-1 was mapped to the X chromosome, 10 centiM away from lon-2 (e678). Viral induced cell fusion and invasion by enveloped viruses are cold-sensitive processes. Many cold-sensitive mutations in different fusogenic viral proteins have been analyzed. Our characterization of duf-1(zu316) is consistent with the idea that membrane fusion is a cold-sensitive process. The future plans are to further characterize the phenotypes at the permissive and restrictive temperatures, find the cold sensitive period and to continue the mapping. 1. Podbilewicz B.(1997) Worm Meeting Abstract, p.300 2. Costa M. and Priess J.(1995) Worm Meeting Abstract, p.165 3. Gattegno T. and Podbilewicz B.(1997) Worm Meeting Abstract, p.546 We thank Mike Costa and Jim Priess for sending zu316 to us.