Table of Contents
Selective cell fusion is a natural part of development. It is found in sexually reproducing organisms that require fertilization to propagate and in muscles, placenta, bones, lens of the eye and stem cells. Cell fusion is particularly important in the development of C. elegans: in addition to 300 sperm and oocytes that fuse during fertilization, 300 of the 1090 somatic cells born, fuse throughout development. Studies of cell fusion in C. elegans have shown that although different types of cells fuse, cell membrane merger is initiated through a common mechanism involving the action of one gene, eff-1. In worms with mutations that inactivate eff-1, almost none of the 300 somatic cells that normally fuse do so, but appear to differentiate, attach and behave in the same way as fusing cells. Such worms develop and survive but have numerous morphological, behavioral and fertility defects associated to cell fusion failure in the epidermis, pharynx, male tail, vulva and uterus. Cell fusion in embryonic dorsal epithelial cells has been analyzed in great detail by confocal microscopy using membrane fluorescent probes, apical junction markers and cytoplasmic aqueous fluorescent probes allowing the direct observation of membrane disappearance, pore expansion and cytoplasmic content mixing. The complete elimination of the membranes between two fusing cells takes about 30 min and involves vesiculation of the fusing membranes. Genetic and cell biological evidence indicates that eff-1 activity is both necessary and sufficient to fuse epithelial and myoepithelial cells in vivo. Based on electron microscopic analyses of intermediates of cell fusion in eff-1 mutants, it appears that eff-1 is required for both initiation and expansion of fusion pores, similar to the fusogen of Influenza virus. While only one gene encoding a novel candidate component of the cell membrane fusion machinery has been found, the nematode’s cell fusion program is under the control of many cell-specific transcriptional regulators. A large number of these conserved regulators prevent cell fusion by repressing eff-1 activity. For example, if either ceh-16/engrailed or the GATA factor EGL-18/ELT-5 is inactivated, the lateral epidermal cells that normally do not fuse in the embryo will fuse causing embryonic lethality. And if either the Hox protein lin-39/Deformed or its cofactor ceh-20/Extradenticle is inactivated, the ventral epidermal vulval precursor cells that normally do not fuse in the larvae will fuse and the hermaphrodite will have no vulva. In addition, there is evidence for coordinated and complex regulation of lin-39 in the ventral epidermis by Ras, Wnt, Rb/E2F, NuRD and lin-15 pathways. It appears that in many cells that normally do not fuse, specific transcription complexes repress eff-1 expression preventing cell fusion. ref-2 (REgulator of Fusion-2) encodes a Zn-finger protein that is required to generate ventral Pn.p cells and to keep them unfused both in males and hermaphrodites. ref-2 is necessary, but not sufficient, to maintain Pn.p cells unfused. This review shows that far from cell fusion being an unusual phenomenon, there is the clear prospect that animal cells in all tissues are intrinsically programmed to fuse, and are only prevented from fusing by transcriptional and post-transcriptional control mechanisms. There are three major questions that remain open for future research: (1) How does eff-1 fuse cells? (2) How do Ras, Wnt, Rb, NuRD, E2F, heterochronic and other pathways control cell fusion? (3) What are the implications of cell fusion beyond worms?
Cell fusion is found in many sexually reproducing organisms that require fertilization or mating to propagate (Heiman and Walter, 2000; Primakoff and Myles, 2002; Shur et al., 2004; Singson et al., 1998; Stein et al., 2004; Trueheart and Fink, 1989; Wassarman et al., 2001; Xu and Sternberg, 2003). Diverse organisms form multinucleate cells in organs such as muscles, placenta, bones, lens of the eye and stem cells, although most cells do not fuse during development. (Baroux et al., 2004; Chen and Olson, 2004 ; Cross et al., 1994; Hickey et al., 2002; Jee and Nolan, 1963; Kuszak et al., 1985; Terada et al., 2002; Xiang et al., 2002; Ying et al., 2002). In addition, cell invasive behaviors involve fusion between cells, such as viral induced cell fusion and anchor cell invasion and fusion in C. elegans (Earp et al., 2005; Newman et al., 1996; Sharma-Kishore et al., 1999). Little is known about the mechanisms of cell fusion. In C. elegans, some of the components of the machinery that brings about and regulates cell fusion have been identified. While many cell-specific transcriptional regulators of cell fusion have been characterized, to date, only one gene encoding a candidate component of the cell membrane fusion machinery has been identified. This chapter includes sections on transcriptional regulation of cell fusion and on a novel candidate cell fusion machinery gene.
Over 30% of all somatic nuclei in C. elegans are contained within giant cells generated by cell fusion (Hedgecock and White, 1985; Podbilewicz and White, 1994; Sulston and Horvitz, 1977; Sulston et al., 1983; White, 1988; see Figure 1; Wormatlas, Epidermal morphogenesis, Male development and Vulval development). These cells are formed during embryonic and larval development in a genetically programmed process. Syncytiogenesis culminates in complex cellular structures, such as, myoepithelial cells in the pharynx and epithelial toroids (ring shape cells) in the uterus, vulva and male tail (Figure 1; Newman et al., 1996; Nguyen et al., 1999; Sharma-Kishore et al., 1999). For reviews on cell fusion in worms see (Podbilewicz, 2000; Podbilewicz and Chernomordik, 2005; Shemer and Podbilewicz, 2000; Shemer and Podbilewicz, 2003; Witze and Rothman, 2002). For sperm-egg fusion see chapters on Control of oocyte meiotic maturation and fertilization and Spermatogenesis.
The largest organ in worms and humans is the epidermis (hypodermis). In some marine nematodes and in vertebrates the skin and most other organs are cellular. However, in C. elegans and related nematodes the epidermis is composed of several distinct multinucleate cells (syncytia; Chitwood and Chitwood, 1974; Hedgecock and White, 1985; Podbilewicz and White, 1994; Sulston and Horvitz, 1977). These syncytia are generated by the fusion of mononucleate cells during development. The largest syncytium is hyp7 which is generated in the embryo during morphogenesis (Figure 1; Podbilewicz and White, 1994; Sulston et al., 1983). During larval development additional cells fuse with hyp7 (Podbilewicz and White, 1994; Yochem et al., 1998). Several cells in the ventral and lateral epidermis remain unfused, divide and generate ectodermal organs including sex-specific mating structures and sensory and motor neurons. Thus, in order for all these organs to be formed, it is essential that the pattern of cell fusions be tightly controlled. To define the spatiotemporal constraints of cell fusion in C. elegans, the order of events in which epithelial cells fuse was studied (Figure 1). To study cell boundaries, apical junctions (AJs) were monitored during fusion using the MH27 monoclonal antibody that recognizes the AJM-1 protein (Francis and Waterston, 1991; Koppen et al., 2001). Using confocal microscopy and 3D reconstructions the sequence of fusions was studied (Figure 2; Podbilewicz and White, 1994). Hypodermal cell fusions start after migration of dorsal epithelial cells. Epidermal fusions usually start in the anterior dorsal epidermis and continue posteriorly. Thus, the fusions in the epidermis are not completely invariant. This is in contrast to the sequence of cell fusions in the vulva that are invariant in C. elegans (Sharma-Kishore et al., 1999). While in other nematode species the vulval cell fusion sequences are different than in C. elegans, they are invariant between animals of the same species (Kolotuev and Podbilewicz, 2004; Louvet-Vallee et al., 2003; See Figure 1 and Figure 2 for more details). The timing and location of postembryonic fusions in the epidermis, vulva, uterus and male tail have been studied in C. elegans and in related nematode species (Figure 2; Newman et al., 1996; Nguyen et al., 1999; Sharma-Kishore et al., 1999). These studies complement the observations of cell fusions obtained by electron microscopy and show intermediates in the sequence of cell fusions during development (Albertson and Thomson, 1976; Newman et al., 1996; Nguyen et al., 1999; Sharma-Kishore et al., 1999; Sulston et al., 1983; White, 1988). See Shemer and Podbilewicz (2000) for a detailed review of cell fusion events.
A major advance in the understanding of developmental cell fusion was the development of a system to reproducibly observe ongoing cell fusions within a live organism. Using multiphoton laser scanning microscopy, it is possible to follow the dynamics of cell membranes during cell fusion with four-dimensional reconstruction of live embryos labeled with the vital fluorescent membrane probe FM-4-64 (Mohler et al., 1998). Syncytiogenesis is followed as the dynamic disappearance of lateral membranes and AJs labeled with AJM-1::GFP (Figure 2A and Figure 3B). Fusion appears to originate close to the apical junction and to expand along the membranes and basally generating an enlarging fusion wave (Figure 3). The complete elimination of the membranes between two fusing cells takes about 30 min (Mohler et al., 1998). The expansion of the fusion zone is accompanied by the migration of the AJ marker AJM-1::GFP from an apical to a basal position (Figure 3C). Disappearance of the basally located AJM-1::GFP protein occurs only 5-10 minutes after the initiation of membrane fusion (Mohler et al., 1998). However, the loss of AJs is not required for all cell fusions since dorsal and ventral cells fuse in the absence of AJs in the contact zone (Podbilewicz and White, 1994) and myoepithelial cells in the pharynx fuse without disassembling the AJs that remain in the apical side of the syncytia (Shemer et al., 2004).
TEM on embryos fixed while fusion events were in progress showed vesiculation of membranes at the fusing zone. The irregular vesicles observed (10–50 nm in diameter) were interpreted as part of a cellular fusion machinery that initiates membrane merger by the formation of a single unique pore at the apex of the fusing cells that expands through radial internalization of the cell membranes (Mohler et al., 1998). However, alternative mechanisms could account for the observations (Podbilewicz and Chernomordik, 2005). Fusion pores can be detected by electrophysiology or by following redistribution of cytoplasmic aqueous probes (e.g., GFP; Mohler et al., 2002; Shemer et al., 2004). However, these methods cannot be used to study the expansion of fusion pores between cells from a few nanometers to micrometers in diameter. The mechanisms responsible for late fusion stages and their dependence on the fusogenic proteins that initiated pore formation remain largely unexplored even in well characterized viral-cell fusion systems (Leikina et al., 2004; Podbilewicz and Chernomordik, 2005). In summary, cell fusion in embryonic dorsal epithelial cells has been analyzed in great detail by confocal microscopy using membrane fluorescent probes (FM4-64; Mohler, 1999; Mohler et al., 1998; Mohler and White, 1998), apical junction markers (AJM-1::GFP; Mohler et al., 1998; Rabin and Podbilewicz, 2000) and cytoplasmic aqueous fluorescent probes (Mohler et al., 2002; Shemer et al., 2004) allowing the direct observation of membrane disappearance, pore expansion and cytoplasmic content mixing. TEM studies of the same cells have shown vesiculation as a possible intermediate in pore expansion (Mohler et al., 1998). Other cell fusions during morphogenesis of the tail showed much larger vesicles but no small irregular vesiculation of the membranes (Nguyen et al., 1999). The vesicles may be recycled to a different domain of the plasma membrane where they can fuse (exocytosis). The overall result of this membrane recycling process may transfer transverse membranes to longitudinal areas allowing efficient elongation of the animal (Podbilewicz, 2000; Shemer and Podbilewicz, 2000). More recently, multiple stable pore intermediates (20–50 nm in diameter) were identified in pharyngeal myoepithelial and epidermal cells in eff-1 mutants (Figure 4; Gattegno et al., unpublished results; Shemer et al., 2004). The emerging picture suggests tissue-specificity in the mechanisms of initiation and expansion of fusion pores in C. elegans offering an amazing system to dissect the stages in the universal and largely unknown process of cell fusion.
Genes encoding fusogenic proteins required for the fusion of enveloped viruses (Earp et al., 2005) and intracellular membrane traffic (See Figure 3A; Jahn et al., 2003) have been identified and characterized. However, developmental cell fusion proteins are responsible for cell attachment before fusion or regulation of cell fusion (Chen and Olson, 2004; Chen and Olson, 2005; Kontani and Rothman, 2005; Shemer and Podbilewicz, 2003; Stein et al., 2004). Over the last decade a few attempts have been made to isolate mutations that specifically block or affect cell fusion in worms (Alper and Kenyon, 2001; Gattegno, 2003; Labouesse, 1997; Mohler et al., 2002; Podbilewicz, 2000; Podbilewicz and Chernomordik, 2005; Shemer and Podbilewicz, 2000; Shemer and Podbilewicz, 2003; Terns et al., 1997). Many successful screens have been designed to isolate spermatogenesis and oogenesis defective mutants that block fertilization (Singson et al., 1998; Xu and Sternberg, 2003). Still, very few mutants are candidates to affect stages after attachment of the two plasma membranes (see Control of oocyte meiotic maturation and fertilization and L'Hernault, 1997; Singson, 2001). Only recently mutations that block most somatic cell fusions in C. elegans were obtained, and they identified one gene that appears to be essential for the merger of the plasma membranes without affecting attachment or other pre-fusion processes (Kontani and Rothman, 2005; Mohler et al., 2002; Gattegno, T., Assaf, N. and BP, unpublished results).
eff-1 encodes a strong candidate developmental fusogen that is expressed in the fusion competent cells prior to cell membrane fusion. Moreover, eff-1 mutant cells are wild-type in events prior to cell membrane fusion. Still the mutant cells fail to fuse and they develop as unfused epithelia within subviable animals containing morphological, behavioral and fertility defects (Cassata et al., 2005; Gattegno, 2003; Mohler et al., 2002; Shemer, 2002; Shemer and Podbilewicz, 2002; Shemer and Podbilewicz, 2003; Shemer et al., 2004; Figure 5). eff-1 is the founder of a novel gene family encoding membrane and secreted proteins conserved within nematodes. No homologous genes have been identified in species outside nematodes (Shemer and Podbilewicz, 2003). Genetic and cell biological evidence indicates that eff-1 activity is both necessary (Mohler et al., 2002) and sufficient (Shemer et al., 2004) to fuse epithelial and myoepithelial cells of the epidermis, vulva, uterus and pharynx in vivo. However, eff-1 is not required in at least one fusion event in worms, the fusion between the anchor cell and the utse cells (Shemer et al., 2004); Anna Newman, personal communication). eff-1 encodes at least four alternatively-spliced isoforms, two predicted type-I membrane glycoproteins (eff-1A and eff-1B) and two secreted proteins (eff-1C and eff-1D; Shemer and Podbilewicz, 2003). eff-1 contains a putative fusion peptide within the protein similar to some class II viral fusogens (Wormbase; Shemer and Podbilewicz, 2003).
Recently, ectopic expression of genomic eff-1A::GFP showed dynamic GFP localization from the cytoplasm to the sites of cell-cell contact, prior to, and during, cell fusion (del Campo et al., 2005). The functions of two extracellular domains of eff-1 that had been hypothesized to have roles in membrane fusion have been tested (Shemer and Podbilewicz, 2003). While transgenic proteins mutated in a putative phospholipase A2 active site rescued a weak eff-1 mutant, two independent mutations in the putative hydrophobic fusion peptide, failed to rescue (del Campo et al., 2005). However, the putative fusion peptide mutated proteins were not expressed on the surface. Thus, the putative fusion peptide is required for correct folding, transport or localization to the site of contact and the direct function of this domain on cell fusion has not been determined (del Campo et al., 2005). It was proposed that homotypic interactions of eff-1A proteins facilitate cell fusion by bringing the membranes into close contact in analogy to SNARE-mediated intracellular fusion (See Figure 3A; del Campo et al., 2005). Since these experiments were done in the background of weak eff-1 mutations, it is conceivable that the ectopic expression of eff-1 protein isoforms can interact and rescue the endogenous partially active eff-1 mutant proteins. Further structure-function analyses of eff-1 isoforms, genetic mosaic analyses and in vitro reconstitution of membrane fusion will determine whether eff-1 alone is sufficient to induce membrane fusion. Nevertheless, it is still possible that eff-1 controls fusion at a post-binding, pre-membrane merger stage. Based on electron microscopic analyses of intermediates of cell fusion in eff-1 mutants, it appears that eff-1 is required for both initiation and expansion of fusion pores (Figure 4), similar to the well characterized fusogen of Influenza virus (Gattegno et al., unpublished results; Leikina et al., 2004; Podbilewicz and Chernomordik, 2005; Shemer et al., 2004).
During morphogenesis the main body epidermis is patterned in three distinct regions with different fusogenic activities (Figure 1; Ding et al., 2004; Michaux et al., 2001; Priess and Hirsh, 1986; Simske and Hardin, 2001): dorsal, lateral and ventral. 1) Two rows of dorsal cells interdigitate forming a single row of cells that fuse forming the hyp6 and hyp7 syncytia as the embryo elongates (Podbilewicz and White, 1994; Priess and Hirsh, 1986; Sulston et al., 1983). 2) Two independent rows of lateral seam cells that elongate but do not fuse during embryogenesis, but their descendants fuse with hyp7, generate neurons or maintain the seam cells during postembryonic development. 3) Two rows of ventral P blast cells that do not fuse in the embryo, but their descendants either migrate and fuse with hyp7 or divide and differentiate into neuronal, vulval, anal or male tail tissues during larval development (Figure 6; Sulston and Horvitz, 1977). Different transcription factors have been identified as regulators of the fusion activities that pattern the epidermis and associated sexual and neuronal organs (Table 1; see also Transcriptional regulation and Transcription mechanisms).
Table 1. Regulators of developmental cell fusion in C. elegans
|Gene||Protein structure and proposed function||References|
|Ventral cell fusions|
|lin-39||Hox (Dfd/Scr)-represses eff-1 expression in hermaphrodites||Clark et al., 1993; Maloof and Kenyon, 1998; Shemer and Podbilewicz, 2002; Wang et al., 1993|
|lin-39||Hox (Dfd/Scr)-inhibits fusion in males when mab-5 is OFF||Clark et al., 1993; Maloof and Kenyon, 1998; Wang et al., 1993|
|lin-39 + mab-5||Hox (Dfd/Scr + Antp) promote fusion in males||Clark et al., 1993; Salser and Kenyon, 1994; Wang et al., 1993|
|mab-5||Hox (Antp) inhibits fusion in males when lin-39 is OFF||Alper and Kenyon, 2001; Kenyon, 1986|
|ceh-20||Hox co-factor (exd/pbx)-represses eff-1 expression||Shemer and Podbilewicz, 2002|
|nhr-25||Ftz-F1/SF1 Hox co-factor-represses fusion with lin-39in P(3-8).p||Chen et al., 2004|
|nhr-25||Ftz-F1/SF1 co-factor-promotes fusion in non-VPCs||Chen et al., 2004|
|acn-1||Angiotensin-converting enzyme-transcriptional target of nhr-25||Brooks et al., 2003; Chen et al., 2004|
|ref-1||bHLH TF (hairy)-inhibits mab-5 activity and lin-39 expression||Alper and Kenyon, 2001|
|egl-27||Chromatin remodelling-regulates Pn.p cell fusion in both sexes||Ch'ng and Kenyon, 1999|
|ref-2||Zinc-finger TF-prevents Pn.p fusion in L1||Alper and Kenyon, 2002|
|egl-18/elt-6||GATA factors-repress cell fusion L2-L4; targets of lin-39/ceh-20||Koh et al., 2004; Koh et al., 2002|
|sem-4||Zinc-finger TF-positively regulates lin-39, preventing fusion||Grant et al., 2000|
|bar-1||Wnt pathway (beta-catenin)-positively regulates lin-39||Eisenmann et al., 1998|
|apr-1||Wnt pathway (APC related factor)-activates bar-1||Hoier et al., 2000|
|let-60||Ras inhibits fusion L1-L3; promotes fusion in late vulva development||Han et al., 1990; Shemer et al., 2000|
|SynMuv class A (e.g., lin-15a)||TFs, NURD complex components and chromatin remodeling-work with class B to antagonize Ras pathway signaling||Chen and Han, 2001; Fay and Han, 2000; Herman and Hedgecock, 1990|
|SynMuv class B (e.g., lin-35/Rb)||TFs, Rb/E2F complex components and chromatin remodeling-work with class A to antagonize Ras pathway signaling promoting fusion||Chen and Han, 2001; Fay and Han, 2000; Herman and Hedgecock, 1990; Myers and Greenwald, 2005|
|SynMuv class C (e.g.,TRRAP)||Tip60/NuA4-like histone acetyltransferase complex and P400 work with class A and B to antagonize Ras pathway signaling||Ceol and Horvitz, 2004|
|fus-1||E subunit of V-ATPase-represses eff-1 activity; late embryogenesis||Kontani et al., 2005|
|Lateral cell fusions|
|nhr-25||Ftz-F1/SF1 TF-promotes cell fusion and/or differentiation||Chen et al., 2004|
|acn-1||Angiotensin-converting enzyme-Molting and morphogenesis||Brooks et al., 2003|
|lin-29||Zinc-finger TF-promotes fusion in L4 molt; heterochronic pathway||Ambros and Moss, 1994; Rougvie and Ambros, 1995|
|egl-18/elt-6||GATA factors-repress cell fusion||Koh and Rothman, 2001|
|ceh-16||Homeobox TF (engrailed)-represses eff-1 expression||Cassata et al., 2005|
|lin-25||Novel protein TF?-overexpression disrupts cell fusion||Nilsson et al., 1998|
|Dorsal cell fusions|
|ceh-13||Hox (labial)-promotes some anterior cell fusions||Brunschwig et al., 1999|
|idf-1||Nd-promotes embryonic cell fusion||Gattegno, 1998; Gattegno, 2003|
|fus-1||E subunit of V-ATPase-represses eff-1activity, late embryogenesis||Kontani et al., 2005|
|Male tip of the tail cell fusions|
|tlp-1||Zinc-finger TF-required for polarity of T lineage and hyp(8-10) fusions||Zhao et al., 2002|
|lep-1||Nd-Required for correct timing of cell fusions and morphogenesis||Nguyen et al., 1999; Zhao et al., 2002|
|Anchor cell-uterine seam cell fusion (AC-utse; vulval-uterine connection)|
|lin-11||LIM -homeobox TF-differentiation of cells that fuse with AC||Newman et al., 1999|
|cog-2/egl-13||Sox-domain TF-maintains fates of cells that fuse with AC||Cinar et al., 2003; Hanna-Rose and Han, 1999|
|smo-1||SUMO-1-Small ubiquitin-like modifier-Modifies lin-11 activity||Broday et al., 2004|
|ty10||sec-18/nsf homolog-intracellular vesicular fusion, promotes AC fusion||A. Newman, personal communication|
|Epithelial and pharyngeal cell fusions but not AC-utse cell fusion|
|eff-1||Novel transmembrane and secreted isoforms-fusion machinery||Mohler et al., 2002; Shemer et al., 2004|
|Identity and putative functions (if mentioned) of genes that control cell fusion during C. elegans development.Class A of the SynMuv genes works redundantly with class B genes to promote fusion. Class C works with classes A and B. Each one of these classes contains several genes. nd- not determined TF-transcription factor. Modified from Shemer and Podbilewicz (2003), Copyright (2003), with permission from Wiley.|
During morphogenesis six ventral cells fuse to 17 dorsal cells to form the large syncytium hyp7 in the embryo. However, 12 P cells covering most of the ventral epidermis do not fuse in the embryo. In the L1, six posterior descendants of the P cells (P(3-8).p; the ventral precursor cells, VPCs) are each competent to generate vulval cells in the hermaphrodite. In the wild-type L3, only the nearest VPCs to the uterine anchor cell (AC) generate the 22 nuclei of the vulva, while the three others fuse to hyp7. The vulval primordial cells divide, migrate and fuse in a stereotyped manner to form an epidermal tube during the L3 and L4 stages (Greenwald, 1997; Kornfeld, 1997; Michaux et al., 2001; Podbilewicz and White, 1994; Sharma-Kishore et al., 1999; Sulston and Horvitz, 1977; Sulston et al., 1983; Wang and Sternberg, 2001). For an overview of ventral cell biology in hermaphrodites and males (see Figure 1 and Figure 6 and the chapters: Epidermal morphogenesis, Hermaphrodite cell-fate specification, Male development, and Vulval development).
The six Hox genes in C. elegans are expressed and determine cell fates along the antero-posterior body axis (Brunschwig et al., 1999; Chisholm, 1991; Clark et al., 1993; Van Auken et al., 2000; Wang et al., 1993; See chapter on Hox genes). In the L1, the Hox genes lin-39/Sex combs reduced/Deformed and mab-5/Antennapedia regulate cell fusion of the Pn.p cells to hyp7 (Figure 6). P(3-8).p cells express lin-39 and P(7-11).p express mab-5 in both sexes (Maloof and Kenyon, 1998; Salser and Kenyon, 1994; Wang et al., 1993). lin-39 prevents fusion of P(3-8).p (vulva precursor cells; VPCs) in the hermaphrodites (Clark et al., 1993; Wang et al., 1993) by repression of eff-1 expression (Shemer and Podbilewicz, 2002). The lin-39 prevention of cell fusion of P(3-8).p is maintained until P(5-7).p cells are induced in the L3 (Maloof and Kenyon, 1998; Shemer and Podbilewicz, 2002). Wnt and Ras signaling control lin-39 activity in the VPCs (Chen and Han, 2001; Eisenmann et al., 1998; Maloof and Kenyon, 1998; see Wnt signaling).
In hermaphrodites mab-5 appears to be largely inactive in P(7-11).p during the L1 cell fusion fate decision (Figure 6C; see below). However, in mab-5 mutant worms, P11.p is sometimes transformed into P12.p (Kenyon, 1986) and in 15% of the mab-5 (lf) hermaphrodites P11.p fails to fuse in the L1 (Alper and Kenyon, 2001). It has been shown that mab-5 has a dynamic and fine control over diverse cellular and morphogenetic activities (Salser et al., 1993). It is possible that mab-5 may also have a complex and dynamic activity in the posterior Pn.p cells during the L3 since some vulval phenotypes have been observed in mab-5 hermaphrodites (Clandinin et al., 1997) and mab-5 mutations in Pristionchus pacificus strongly affect vulva formation (Gutierrez et al., 2003; Jungblut et al., 2001; Jungblut and Sommer, 1998; Ira Kolotuev and BP, unpublished results; see Evolution of development in nematodes related to C. elegans).
eff-1 is also epistatic to ceh-20, encoding a known cofactor of lin-39 (Koh et al., 2002; Liu and Fire, 2000). ceh-20/Extradenticle may act in a complex with lin-39 and other cofactors to repress eff-1 transcription (Shemer and Podbilewicz, 2002). nhr-25/Ftz-F1/SF1 may also act as a cofactor of lin-39 during regulation of fusion in the Pn.p cells (Asahina et al., 2000; Chen et al., 2004; Gissendanner and Sluder, 2000). nhr-25 may act with lin-39 to repress fusion of P(3-8).p while acting with another transcription factor in neighboring cells to promote cell fusion and cell shape dynamic changes (Chen et al., 2004; Silhankova et al., 2005). A potential transcriptional target of nhr-25 is acn-1, a gene encoding a member of the angiotensin-converting enzyme family (Brooks et al., 2003; Chen et al., 2004).
The following working model is emerging for the regulation of cell cycle and cell fusion effectors by Hox proteins and cofactors in the ventral epidermis. First, a complex of lin-39/ceh-20 (Koh et al., 2002; Liu and Fire, 2000) together with other transcription factors and cofactors represses eff-1-mediated plasma membrane fusion during competence and induction in the L1 and L3 respectively. Second, lin-39 also stimulates cell division. Third, VPCs that failed to fuse and to divide in lin-39;eff-1 double mutants can perform other cellular behaviors characteristic of their normal great-granddaughters, such as formation of vulval rings (Shemer and Podbilewicz, 2002) and partial Mid-L4 expression of vulval markers like egl-17::GFP (Burdine et al., 1998; Cui and Han, 2003). Thus, in the absence of lin-39 activity, induction of the VPCs is independent of their patterns of cell division (sublineages) and a double block in cell fusion and cell cycle can allow cell differentiation (Shemer and Podbilewicz, 2002). Cell fusion inhibition is not sufficient for the VPCs to form a vulva because lin-39 is also required for these cells to proliferate. Although undivided VPCs do form vulval rings, these are non-functional (Shemer and Podbilewicz, 2002). Finally, during late stages in vulva morphogenesis, Ras appears to have a cell fusion promoting function (Shemer et al., 2000).
Two transcription factors, egl-27 and ref-1, maintain mab-5 inactive in the hermaphrodite. In a screen for mutations that modify the pattern of Pn.p cell fusion, ref-1 (mu220; REgulator of Fusion-1) was isolated. ref-1 mutation affects Pn.p cell fusion early but not later during larval development (Figure 7). ref-1, a protein with two bHLH domains, allows fusion of posterior Pn.p cells in the hermaphrodites by inhibiting the sex-specific mab-5 activity (Alper and Kenyon, 2001). In addition, ref-1 functions together with mab-5 to prevent lin-39 expression in the posterior region allowing fusion of posterior Pn.p cells. The third action of ref-1 is independent of mab-5, ref-1 can influence the Hox gene egl-5’s ability to regulate Pn.p cell fusion (Alper and Kenyon, 2001). egl-27, a chromatin regulatory factor (Ch'ng and Kenyon, 1999; Herman et al., 1999; Solari et al., 1999), regulates Pn.p cell fusion in both sexes. In egl-27 mutant hermaphrodites, posterior Pn.p cells fail to fuse. egl-27 regulates expression of lin-39, mab-5 and egl-5 controlling fusion of specific ventral cells. In males, mab-5 and lin-39 inhibit each other’s activity when expressed in the same Pn.p cell, resulting in fusion of these cells (See Figure 6D). In males, egl-27 permits the combinatorial interaction between lin-39 and mab-5. Thus, egl-27 activation appears to provide cell- and sex- specific regulation of Hox function that creates antero-posterior patterns of cell fusion (Ch'ng and Kenyon, 1999). egl-27 and ref-1 could directly, or indirectly through Hox and GATA factors and other cofactors, control transcription of eff-1 along the antero-posterior axis to generate a complex pattern of Pn.p cell fusion (see below).
In males, lin-39 and mab-5 proteins inhibit each other even when one of the Hox proteins is ectopically over-expressed relative to the other (Salser et al., 1993). Alper and Kenyon hypothesized that both Hox proteins may bind to regulatory sites in a cell fusion gene encoding a direct effector of cell fusion (Alper and Kenyon, 2002). In their model, the binding of either Hox protein alone would inhibit expression of a fusion gene and expression of both Hox proteins would promote expression of a fusogen resulting in Pn.p cell fusion. Alternatively, the two Hox proteins together could repress a cell fusion inhibitor (ref-2; REgulator of Fusion-2) required to prevent expression of a cell fusion gene promoting cell fusion (Figure 8; Alper and Kenyon, 2002). To investigate this complex sex-specific regulation of Pn.p cell fusion, the dominant ref-2 (mu218) mutation was isolated and cloned (Figure 9A; Alper and Kenyon, 2002). ref-2 encodes a Zn-finger protein of the odd-paired (opa)/Zic family and is required to generate Pn.p cells and to keep them unfused both in males and hermaphrodites. ref-2 is necessary, but not sufficient, to maintain Pn.p cells unfused. In addition, an active Hox gene is also necessary to prevent fusion (Figure 8–Figure 10).
In summary, either mab-5 or lin-39 act with ref-2 to inhibit Pn.p cell fusion in males. When mab-5 and lin-39 are both active, fusion of Pn.p cells is not inhibited, perhaps due to low ref-2 expression. In hermaphrodites, egl-27 and ref-1 prevent mab-5 from affecting Pn.p cell fusion and they may act by preventing mab-5 activity on ref-2 expression. It appears that eff-1 is the ultimate effector of cell fusion in the ventral epidermis whose complex expression must be finely regulated through Hox, ref-1, ref-2 and other interacting genes in both sexes (Figure 8).
ELT-5/EGL-18 and ELT-6 GATA factors repress cell fusion in the VPCs (Figure 11; Koh et al., 2004; Koh et al., 2002). The cell fusion repression in the ventral epidermis appears to be controlled by lin-39/ceh-20 heterodimers binding to two HOX/PBC sites in a vulval enhancer of egl-18/elt-6. Thus EGL-18/ELT-5 and ELT-6 are genetically redundant regulators of cell fusion in the vulva primordium and may be direct targets of lin-39 (Koh et al., 2002). Moreover, reduction of lin-39 activity resulted in lower elt-6::GFP expression and over-expression of egl-18 is sufficient to partially rescue vulval defects in lin-39 (RNAi), consistent with the model in which egl-18/elt-5 and elt-6 act downstream of lin-39. It has been proposed that these GATA factors repress genes that promote Pn.p cell fusion, such as eff-1 (Koh et al., 2004; Koh et al., 2002; see below) and also promote cell proliferation (Koh et al., 2004). One speculation is that EGL-18 and ELT-5 may also act in desilencing of condensed chromatin in the developing vulva (Koh et al., 2004). In addition, SEM-4 Zn finger transcription factor promotes vulval cell fates through lin-39 (Eisenmann and Kim, 2000; Grant et al., 2000).
SynMuv genes and in particular Rb/E2F and NuRD (nucleosome remodeling and histone deacetylation) complexes regulate lin-39 expression and related activities during Pn.p fusion, competence and inductions (Ch'ng and Kenyon, 1999; Chen and Han, 2001; Chen and Han, 2001; Herman et al., 1999; Solari and Ahringer, 2000; Solari et al., 1999). While the NuRD complex negatively regulates lin-39 activity, Rb/E2F appears to have a negative role controlling lin-39 activity and a positive role regulating its expression (Chen and Han, 2001). These apparently complex genetic interactions may not be as a result of direct transcriptional regulation in all cases because some SynMuv genes do not function in the VPCs where lin-39 activity is expressed. In particular, mosaic analyses have shown that lin-15 (Herman and Hedgecock, 1990), lin-37 (Hedgecock and Herman, 1995) and lin-35/Rb (Myers and Greenwald, 2005) act in the hyp7 hypodermal syncytium and not in the VPCs to inhibit vulval fates. Thus, these results are inconsistent with the view that Rb, E2F, histone deacetylases and components of the NuRD and other chromatin remodeling complexes (Ceol and Horvitz, 2001; Ceol and Horvitz, 2004; Chen and Han, 2001; Chen and Han, 2001; Lu and Horvitz, 1998; Solari and Ahringer, 2000; Thomas et al., 2003; von Zelewsky et al., 2000) antagonize the inductive signaling pathways only by acting in the VPCs to transcriptionally repress effector genes required for vulval cell fates. lin-15, lin-35, lin-37 and probably other SynMuv genes may repress vulval fates by regulating a negative signal from hyp7 to the VPCs or by promoting cell fusion probably through activation of a cell fusion effector gene such as eff-1 (Herman and Hedgecock, 1990; Myers and Greenwald, 2005).
In summary, there is evidence for coordinated and complex regulation of lin-39 in the ventral epidermis by Ras, Wnt and SynMuv pathways (Chen and Han, 2001; Eisenmann et al., 1998; Fay and Han, 2000; Gleason et al., 2002; Hoier et al., 2000; Lu and Horvitz, 1998). In addition, the SEM-4 transcription factor (Eisenmann and Kim, 2000; Grant et al., 2000) and GATA factors (Koh et al., 2004; Koh et al., 2002) regulate lin-39. Overall, there appears to be several signaling pathways that control the ability of each of the ventral epidermal cells to fuse, or not to fuse, to hyp7.
Lateral seam cells actively participate in embryonic elongation but do not fuse in the wild-type embryo. However, during postembryonic development the ectoblastic seams have dynamic cell behaviors essential for the growth of hyp7 by cell fusion, generation of neurons, molting and larval elongation (Hedgecock and White, 1985; Podbilewicz and White, 1994; Singh and Sulston, 1978; Sulston and Horvitz, 1977).
It has recently been shown that ceh-16/engrailed is expressed in the lateral seam cells during embryonic development and induces the expression of seam cell markers including ELT-5 (Cassata et al., 2005). In contrast, ceh-16 represses eff-1 expression in the seam cells preventing these cells from fusing (Figure 12).
In ceh-16 mutants and ceh-16 (RNAi) worms, eff-1 is ectopically expressed in the seam cells resulting in cell fusion, morphogenesis block and embryonic lethality (Figure 13). In addition, ceh-16 mutant seam cells migrate dorsally or ventrally losing their lateral position. Animals expressing ceh-16 in some but not all seam cells in eff-1(-) background had a complete fusion failure suggesting that eff-1 acts downstream of ceh-16 (Figure 13). Thus, ceh-16 appears to maintain embryonic epidermal compartments by preventing cell fusion and maintaining the lateral seam cell borders that separate the dorsal and ventral hypodermal compartments during embryogenesis (Figure 12D; Cassata et al., 2005).
Two GATA factors, ELT-5/EGL-18 and ELT-6 (see above), are critical for the differentiation and repression of cell fusion of the lateral seam cells (Figure 14; Koh and Rothman, 2001). Since in elt-5 (RNAi) animals the seam cells migrate and fuse ectopically (Koh and Rothman, 2001), ceh-16 may act indirectly by activating elt-5, which in turn represses eff-1. This is supported by inappropriate expression of eff-1 p::gfp in elt-5 (RNAi) embryos (Figure 15; Cassata et al., 2005). Since elt-5/elt-6 and ceh-16 are necessary to maintain the identity of the seam cells, the repression of eff-1-mediated cell fusion is probably only one of the functions of these transcription factors (Figure 12).
Interestingly, cell fusion is also repressed in the ventral region of the epidermis by expression of other homeobox containing proteins lin-39/Dfd and ceh-20/Exd (Shemer and Podbilewicz, 2002) acting also through ELT-5 (Koh et al., 2002; see above). Thus, expression of distinct homeobox containing proteins in lateral and ventral hypodermal compartments may regulate cell fates through ELT-5-mediated repression of eff-1 during embryonic and larval development (Figure 12D). While Wnt and Ras signaling pathways control the expression of lin-39 in VPCs (Eisenmann and Kim, 2000; Eisenmann et al., 1998; Gleason et al., 2002; Maloof and Kenyon, 1998), it is not known what signaling pathways control ceh-16 activity in the seam cells (Cassata et al., 2005). Very few effectors of Hox genes and engrailed have been identified in any organism. In C. elegans, a putative cell fusion protein (eff-1) is a strong candidate to be an effector of ELT-5/6, lin-39-ceh-20/Extradenticle and ceh-16/engrailed in distinct developmental regions and times (Cassata et al., 2005; Shemer and Podbilewicz, 2002).
The timing of larval development is regulated by the expression of micro RNAs (Ambros, 2004; See C. elegans microRNAs). Expression of the lin-4 micro RNA during the L1 is required for down-regulation of other heterochronic genes (e.g. lin-14) and execution of L2 developmental programs (Ambros and Horvitz, 1987; Lee et al., 1993). Likewise, expression of the let-7 micro RNA during the L4 controls the levels of other heterochronic genes (e.g. lin-29) required for adult developmental programs (Reinhart et al., 2000). Mutations in heterochronic genes result in precocious or retarded phenotypes that affect the timing of cell fusion events throughout larval development. lin-29 controls late fusion events in the L4 larvae, including the fusion between seam cells during the last molt (Ambros, 1989; Ambros and Horvitz, 1984; Slack and Ruvkun, 1997). lin-29 regulates the timing of late gene expression during organogenesis of the lateral epidermis, vulva, uterus and male reproductive system (Bettinger et al., 1997; Bettinger et al., 1996; Euling et al., 1999; Newman et al., 2000; Rougvie and Ambros, 1995). lin-29 activities are required for seam cells-vulval-uterine connections involving cell fusion events in the three interacting organs (Newman et al., 2000). Thus, lin-29 controls cell cycle exit, secretion of adult cuticle and cell fusion. It is not known whether eff-1 is an effector of lin-29 and the heterochronic pathway.
Dorsal hypodermal cells fuse during embryogenesis and most of the dorsal cells complete the fusion process halfway through morphogenesis. Very few genes controlling dorsal cell fusions have been identified. The Hox gene ceh-13 controls the embryonic fusion of some anterior dorsal hypodermal cells (Brunschwig et al., 1999; Wittmann et al., 1997) and the Hox cofactor nhr-25 (see above) may also affect the most anterior dorsal cell fusion in hyp7 (Chen et al., 2004).
idf-1 (zu316), an embryonic lethal mutation isolated in a screen for elongation defective embryos (M. Costa and J. Priess, personal communication), is characterized by a block in embryonic dorsal cell fusion. While the idf-1 (Irregular Dorsal Fusion-1) phenotype is variable, all the dorsal cells can fail to fuse. Irregular cell fusion events occurring in idf-1 mutants are retarded compared to wild-type. The anterior dorsal cells have a higher probability of remaining unfused than posterior cells. However, all dorsal cells are able to remain unfused in the mutant, suggesting that idf-1 is required as part of the fusion machinery or in its regulation and not only as a regional regulator of the process (T. Gattegno and BP, unpublished results; Gattegno, 1998; Gattegno, 2003; Gattegno et al., unpublished results).
fus-1 gene protects dorsal and ventral cells from late embryonic cell fusions. In strong fus-1 mutations (Terns et al., 1997), all epidermal cells fuse except the seam cells. eff-1 is epistatic to fus-1. fus-1 encodes a membrane e subunit of the vacuolar H+-ATPase localized to the apical membranes of epidermal fusogenic cells. Thus, eff-1 may be mislocalized to cell membranes in the absence of V-ATPase activity and/or expression, resulting in ectopic hyperfusion (Kontani et al., 2005). The V-ATPase appears to be the first posttranscriptional repressor of eff-1-mediated cell fusion.
The utse is a H-shaped cell that connects the uterus to the seams with a thin hymen separating the vulval and uterine lumens. This syncytium is formed in the late L4 stage by cell fusion of the AC and 8 out of 12 π daughters (Newman and Sternberg, 1996; Newman et al., 1996; Sharma-Kishore et al., 1999). The cog-2/egl-13 SOX domain and the lin-11 LIM domain transcription factors are expressed in π cells and their daughters (Cinar et al., 2003; Hanna-Rose and Han, 1999; Newman et al., 1999). In lin-11 mutants, the AC fails to fuse with the utse and a functional uterine-vulval connection is not formed (Newman et al., 1999). In addition, lin-11 is covalently bound to SUMO (smo-1) and this modification is required for AC-utse and intratoroidal vulval fusions (Broday et al., 2004). In egl-13 mutants, the AC also fails to fuse to the utse (Cinar et al., 2003; Hanna-Rose and Han, 1999) because the π cell lineage is initiated but not completed correctly (Cinar et al., 2003). The disruption of π cell development is at an earlier stage in egl-13 than in lin-11 (Cinar et al., 2003). lin-11 appears to be a target of LIN-12 signaling in the π cells (Gupta and Sternberg, 2002). Thus, both lin-11 and egl-13/cog-2 are required for the maintenance of π cell fate and the differentiation of its progeny and not directly for cell fusion of the AC to utse. In addition, this heterotypic cell fusion event has been shown to be eff-1-independent (Shemer et al., 2004), and a NEM-sensitive factor (NSF/SEC-18) appears to be required for the AC-utse cell fusion and the formation of the uterine-vulval connection (A. Newman, personal communication).
C. elegans has become an excellent research system to study cell membrane fusion and to understand the functions, regulation and mechanisms of cell fusion. Mutations in eff-1 result in a complete and specific block of cell fusion without affecting other cellular functions like cell migration and adhesion. While the mechanisms of cell fusion are poorly understood, it is already clear that eff-1 has a central role in most cell fusions in C. elegans. A working model is that stochastic fluctuations in the levels of eff-1 on the plasma membranes, coupled to transcriptional and post-transcriptional mechanisms that amplify these changes, may lead to fusion of some cells where the levels of activated eff-1 reach a threshold that triggers membrane fusion (Figure 16). A large number of regulatory genes and many more still to be discovered control the formation of a giant multinucleate cell. Most of these genes appear to precisely regulate eff-1 transcription throughout development. It is expected that numerous posttranslational regulators of cell fusion will also act on eff-1 stability and localization. In particular, it appears that in the worm and probably in other organisms as well, there is a widespread repression of cell membrane fusion and not only specific activation of this process. This is probably due to the negative effects of hyperfusion on viability. As occurs in program cell death in worms, ectopic activation of cell fusion results in embryonic lethality, while cell fusion failure results in sub viable larvae and adults with many morphological and behavioral defects. Many fascinating questions are completely open and waiting for a critical mass of C. elegans biologists interested in answering them.
|Is there a trigger (e.g. binding, low pH, calcium) that initiates cell fusion?|
|What are the intermediates and detailed kinetics of early and late fusion steps?|
|What is the fate of the vesicles generated during cell fusion?|
|How does eff-1 affect intracellular membrane dynamics (see Figure 3 and Figure 18)?|
|How does eff-1 fuse cells?|
|What domains of the eff-1 protein have central roles in cell fusion?|
|Which eff-1 isoform(s) are essential and sufficient for cell fusion?|
|Is eff-1 a fusogen similar to viral class I or II fusogens or is it a class of its own?|
|What is the structure of eff-1?|
|Is there cooperativity between different eff-1 proteins to fuse cells?|
|Is eff-1 the only effector of cell fusion in C. elegans?|
|Are there eff-1 receptors and co receptors?|
|Does eff-1 have functions independent of cell fusion?|
|Does eff-1 act as an anti-cell migration factor in the epidermis (see Figure 17 and Figure 18)?|
|Does cell fusion have a role in larval elongation?|
|Are the late defects that develop after fusion failure direct or indirect?|
|Are there other genes required for cell attachment, pore formation, expansion of cytoplasmic bridge and rearrangement of cytoplasmic contents during cell fusion?|
eff-1 is controlled by regulators of cell fusion (ref-1 and ref-2), Hox genes and other cofactors in the ventral Pn.p cells and by the homeodomain gene ceh-16/engrailed and GATA factors ELT-5/EGL-18 in the lateral seam cells.
|Are these regulators acting directly or indirectly on eff-1 transcription?|
|Are there posttranscriptional mechanisms of regulation of eff-1?|
|How does a V-ATPase represses eff-1-mediated epidermal fusion?|
|How does the heterochronic pathway control cell fusion?|
|How are dorsal hypodermal cell fusions regulated in the embryo?|
Cell fusion is a very frequent and naturally occurring process about which very little is known. We are beginning to understand why cells fuse during the formation of organs in C. elegans (Figure 5). Cell fusion research has potential applications in the fields of contraception, cancer, gene therapy and stem cell research. Cell fusions in the epidermis and vulva of C. elegans and during the formation of muscles, bones and placenta in humans, involve regulation of cell differentiation. Dividing cells that are potentially invasive (e.g. epidermal, muscle, placenta and stem cells) fuse to form part of multinucleated cells, stop dividing, and become differentiated. Thus, defects in cell fusion during development may result in cancerous growth in mammals (Duelli and Lazebnik, 2003). However, as with apoptosis, this is not the case in worms. Finally, laser-induced cell fusion in worms (Irle and Schierenberg, 2002; Schierenberg, 1984; Schierenberg, 1987) and other organisms (Avigan et al., 2004; Lundqvist et al., 2004) is an under-explored technique with great potential for understanding many basic and applied biological questions.
I thank Scott Alper, Jim Kramer and Gidi Shemer for critically reading the manuscript. I thank past and present members of the worm community in Haifa for invaluable contributions and members of the Chernomordik lab and the worm community of Bethesda for their support. Supported by grants from the Israel Science Foundation, Fund for the Promotion of Research at the Technion and HFSP.
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*Edited by James M. Kramer and Donald G. Moerman. Last revised April 28, 2005. Published January 06, 2006. This chapter should be cited as: Podbilewicz, B. Cell fusion (January 06, 2006), WormBook, ed. The C. elegans Research Community, WormBook, doi/10.1895/wormbook.1.52.1, http://www.wormbook.org.
Copyright: © 2006 Benjamin Podbilewicz. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.