Epithelial Biology: Lessons from Caenorhabditis Elegans

Home , Alae (nematode anatomy), Caenorhabditis elegans, Septate junction

Gene 277 (2001) 83–100 www.elsevier.com/locate/gene Review Epithelial biology: lessons from Caenorhabditis elegans

Gre´goire Michaux, Renaud Legouis1, Michel Labouesse*

Institut de Ge´ne´tique et de Biologie Mole´culaire et Cellulaire, CNRS /INSERM /ULP, BP. 163, F-67404 Illkirch Cedex, C.U. de Strasbourg, Strasbourg, France Received 4 May 2001; received in revised form 17 August 2001; accepted 4 September 2001 Received by A.J. van Wijnen

Abstract Epithelial cells are essential and abundant in all multicellular animals where their dynamic cell shape changes orchestrate morphogenesis of the embryo and individual organs. Genetic analysis in the simple nematode Caenorhabditis elegans provides some clues to the mechanisms that are involved in specifying epithelial cell fates and in controlling specific epithelial processes such as junction assembly, trafficking or cell fusion and cell adhesion. Here we review recent findings concerning C. elegans epithelial cells, focusing in particular on epithelial polarity, and transcriptional control. q 2001 Elsevier Science B.V. All rights reserved.

Keywords: Caenorhabditis elegans; Epithelial cell; Differentiation; Morphogenesis; Adherens junction; Hemidesmosome; Extracellular matrix; Trafﬁcking; Cell fusion; Transcription

1. Introduction transcription factors control the onset of ‘epithelialisation’ by switching on the expression of proteins that establish cell Epithelial cells play an essential role during development polarity. Similarly, we do not know, except in a few cases and adult life by shaping organs, and by acting as a selective such as branching of the tracheal system (Metzger and Kras- barrier to regulate the exchange of ions, growth factors or now, 1999) and dorsal closure in Drosophila (Noselli and nutrients coming from the outside environment (Yeaman et Agnes, 1999), which genes induce major morphogenetic al., 1999). These functions rely on proper cell fate specifica- processes. In addition, the detailed genetic analysis of the tion and on the acquisition of a polarised phenotype. Epithe- mechanisms underlying morphogenesis is a relatively new lial cells can also easily become cancerous and a direct link area, and what we know comes mainly from the fly. between tumorigenesis, loss of cell polarity and adhesion has The nematode Caenorhabditis elegans provides another long been noted (Thiery et al., 1988; Behrens et al., 1989). powerful system to address the questions raised above Despite their importance and widespread occurrence, using a genetic approach in the integrated context of a many aspects concerning the biology of epithelial cells are live animal. Here we highlight recent progress in the analy- not fully understood. For instance, we do not know if specific sis of different aspects of C. elegans epithelial biology. We first discuss specification of epithelial cell fates in the Abbreviations: AC, Anchor Cell; ADAM, A Disintegrin And Metallo- embryo (Section 3). Then we review how apico-basal proteinase; AJ, Adherens Junction; CeAJ, C. elegans Apical Junction; polarity is established and maintained (Sections 4 and 7), DTC, Distal Tip Cell; DU, Dorsal Uterine (precursor); EGF, Epidermal how the epidermis becomes connected to the underlying Growth Factor; FNIII, FibroNectin type III; GFP, Green Fluorescent muscles (Section 5), and how the basal lamina influences Protein; GPI, GlycoPhosphoInositol; GST, Glutathione S-Transferase; IF, morphogenesis of internal epithelia (gonad, intestine and Intermediate Filament; LC, Linker Cell; LRR, Leucine Rich Repeat; MAGUK, Membrane-Associated Guanylate Kinase; MAP, Mitogen Acti- pharynx) (Sections 6, 8 and 9). Section 10 is devoted to vating Protein; MDCK, Madin–Darby Canine Kidney; PDZ, PSD95/Dlg/ pattern formation and morphogenesis of the egg-laying ZO-1; SJ, Septate Junction; sujn, spermatheca uterine junction (uterine system (uterus and vulva). valve); TJ, Tight Junction; ut, uterine toroid; utse, uterine-seam (cell); uv, uterine vulva (cell); VPC, Vulval Precursor Cell; VU, Ventral Uterine (precursor) 2. Background anatomy * Corresponding author. Tel.: 133-3-8865-3393; fax: 133-3-8865-3201. E-mail address: [email protected] (M. Labouesse). 1 Present address: CGM-CNRS, Avenue de la Terrasse, 91198 Gif/Yvette There are three broad categories of epithelial cells in C. Cedex, France. elegans: those that contribute to a classical epithelium by

Table 1 Phenotypes associated with some genes acting in epithelial cellsa

Tissue Process Gene Phenotype Refs.

Animal Cell

Major epidermis Specification elt-1 Emb lethal Absent 1 Differentiation lin-26, elt-5/6 Emb/larval lethal Ab. cells/defective elongation 2–5 Junction formation let-413, dlg-1, ajm-1 Emb lethal No elongation/ab. junctions 6–9 Morphogenesis hmr-1/hmp-1/hmp-2, let-502 Emb lethal No enclosure and/or no elongation 10–12 Connection with muscles mup-4, mua-3, let-805 Emb/larval lethal No muscle anchoring/ab 13–17 hemidesmosomes Trafficking che-14, lrp-1 Part larval lethal Cuticle defects 18,19 Vulva Specification Ras/MAPK pathway Egl Vul or Muv 20 Morphogenesis sqv-1, sqv-7, sqv-8 Egl or Ste Squashed vulva 21,22 Vulva/Uterus Connection cog-2, lin-11 Egl No connection 23,24 Intestine Specification EDR (end-1 1 end-3) Emb lethal* Absent 25–27 Differentiation elt-2 Larval lethal Ab. cells/no food intake 28 Morphogenesis lin-12/glp-1 pathway Emb lethal# No rotation 29

a This table provides a few examples of the terminal phenotypes associated with mutations affecting epithelial cells but is not an exhaustive list of all genes acting in epithelia. Emb: embryonic; Egl: egg-laying defective; Ste: sterile; Vul: vulvaless; Muv: multivulva; Ab: abnormal; Part: partial; EDR: endoderm determining region (so far only large chromosomal deficiencies removing both end-1 and end-3 lead to absence of the intestine; the intestinal phenotype of those deficiencies can be rescued by introduction of an end-1 or end-3 transgene); MAPK: mitogen associated protein kinase. *Lethality associated with deficiencies removing the EDR are probably linked to the size of the deficiency. #Mutants in the lin-12/glp-1 pathway affect many other processes which contribute to embryonic lethality. 1 (Page et al., 1997); 2 (Koh and Rothman, 2001); 3 (Labouesse et al., 1994); (Labouesse et al., 1996); 5 (Quintin et al., 2001); 6 (Bossinger et al., 2001); 7 (Koeppen et al., 2001); 8 (Legouis et al., 2000); 9 (McMahon et al., 2001); 10 (Costa et al., 1998); 11 (Raich et al., 1999); 12 (Wissmann et al., 1997); 13 (Bercher et al., 2001); 14 (Gatewood and Bucher, 1997); 15 (Hong et al., 2001); 16 (Hresko et al., 1999); 17 (Plenefisch et al., 2000); 18 (Michaux et al., 2000); 19 (Yochem et al., 1999); 20 (Sternberg and Han, 1998); 21 (Herman and Horvitz, 1999); 22 (Herman et al., 1999); 23 (Hanna-Rose and Han, 1999); 24 (Newman et al., 1999); 25 (Zhu et al., 1997); 26 (Zhu et al., 1998); 27 (Maduro et al., 2001); 28 (Fukushige et al., 1998); 29 (Hermann et al., 2000). forming either a sheet (i.e. the epidermis and vulva) or a principally on the first category, particularly on epidermal tube (i.e. the intestine and pharynx); those that are isolated cells which have been well-characterised in nematodes (i.e. the excretory cell); those that form a contractile myoe- (Fig. 1A). Epidermal cells are often called hypodermal pithelial organ (i.e. the spermatheca) (White, 1988). Muta- because they secrete a collageneous cuticle at their apical tions that affect the differentiation, structure, surface, which acts as an exoskeleton to maintain the shape morphogenesis or function of epithelial cells generally of the animal and to anchor muscles. Three classes of result in lethality (see Table 1). This review will focus epidermal cells can be distinguished: cells which cover

Fig. 1. Epithelial cells in Caenorhabditis elegans. (A) Schematic drawing illustrating the relative positions and sizes of the four main epithelial tissues that will be discussed in this review, at mid-embryogenesis (1.5-fold embryo) and in the adult. (B) Confocal picture of an embryo showing internal epithelia (pharynx and intestine) stained with antibodies against PAR-3 (green) which marks the lumen, and the MH27 monoclonal antibody against AJM-1 (red) which marks junctions (the inset on the right corresponds to an enlargement of the boxed area). (C) Confocal picture of a young transgenic larva showing internal epithelia (pharynx and intestine) expressing a DLG-1::GFP translational fusion. Anterior to the left and dorsal up in A-B; anterior in the right-hand corner in C. (D) In the epidermis (simplified diagram on the left), confocal and electron microscopy analyzes have suggested that junctions (example on the right) comprise two independent units (McMahon et al., 2001). The apical-most unit, which is critical to anchor the actin cytoskeleton, corresponds to the classical cadherin-catenin complex encoded by the genes hmr-1 (E-cadherin), hmp-2 (b-catenin) and hmp-1 (a-catenin). The other unit corresponds to a complex formed by the MAGUK protein DLG-1, AJM-1 and possibly other proteins. The electron dense structure visible by electron microscopy (enlarged in pink inset) coincides at least with the AJM-1/DLG-1 unit, and might even be restricted to it. Eliminating the function of a gene in one unit appears to leave the other unit largely unaffected. There are other known proteins at the CeAJ, such as the APC-related protein APR-1 which is essential for the process of ventral enclosure (see Fig. 3A) (Hoier et al., 2000) or small GTPases (Chen and Lim, 1994), however it is not known whether these protein colocalise with HMP-1 or with AJM-1. LET-413 is a basolateral protein required to coalesce both units at a sub-apical position during epidermal differentiation. (E) Muscle cells are tightly connected across the epidermis to the external cuticle, resulting in a strong flattening of the epidermis in areas of muscle contact (diagram on the left). Sarcomeres are anchored to the basal lamina through a structure called the dense body (thick black box), and are connected to fibrous organelles in the epidermis. Fibrous organelles are made up of two substructures, both of which are morphologically similar to vertebrate hemidesmosomes. The molecular composition of each substructure on the apical and basal side of the epidermis is only partially known and probably includes unique and common proteins, such as the protein recognised by the monoclonal antibody MH5 (Francis and Waterston, 1991). The apical substructure, which attaches the epidermis and muscle to the cuticle, contains the proteins MUP-4 and MUA-3, while the basal one, which links the epidermis to the basal lamina, contains myotactin (see Section 5). Intermediate filaments (IFs) connect both substructures. The micrograph on the right shows an area of muscle-epidermis contact in a late embryo, where the cuticle, fibrous organelles, the flattened out epidermis, dense bodies and sarcomeres can be recognised (dense bodies and sarcomeres are still poorly developed compared to larvae). Scale bar 10 mm (B, C), 300 nm (D, E). G. Michaux et al. / Gene 277 (2001) 83–100 85 the body and form the so-called major epidermis; small of excellent recent reviews (Chin-Sang and Chisholm, specialised epidermal syncytia which cover the head and 2000; Hubbard and Greenstein, 2000; Simske and Hardin, the tail; and interfacial epidermal cells which connect inter- 2001). nal organs to the epidermis generally by adopting a toroidal structure as in the rectum and the vulva (White, 1988). The morphogenetic events that allow a functional vulval-uter- 3. Specification of epithelial cell fates in the embryo ine connection to be made have recently been thoroughly investigated and will be discussed in detail. Intestinal cells The pathways involved in specifying epithelial cell fates represent also extensively studied epithelial cells; we will differ in detail between various cell types, but are quite simi- describe the morphogenetic events that lead to an epithelial lar in a more global sense, at least in the embryo (for an earlier tube. Due to space constraints, we will not deal with the review, see Labouesse and Mango, 1999). We will concen- development of isolated epithelial cells. Similarly, we will trate mostly on late aspects of epidermal differentiation. only briefly mention morphogenesis of the embryo and of A model for the specification of the intestine has the somatic gonad, since these subjects have been the topic recently been suggested and can be taken as a paradigm 86 G. Michaux et al. / Gene 277 (2001) 83–100

Fig. 2. Cell fate specification in the epidermis and intestine. Four distinct levels of regulation are involved in specifying intestinal cell fates. (Left) In response to a Wnt and a MAP kinase pathway, the GATA factors MED-1 and MED-2, perhaps with the bZIP transcription factor SKN-1, induce expression of the GATA factors END-1 and END-3, which redundantly act to specify and maintain intestine identity. END-1/3 proteins then activate expression of ELT-2, another GATA factor that turns on expression of different terminal differentiation factors such as the junction component AJM-1, the gut esterase GES-1, or the epitope recognised by the monoclonal antibody MH33. PHA-4, which specifies pharynx identity, is also expressed in the intestine where its specific function is not known. (Right) In lineages generating the major epidermal cells, the GATA factor ELT-1 specifies epidermal identity. Its most likely targets are genes encoding the atypical C2H2 zinc-finger protein LIN-26 which is required for epithelial differentiation, the GATA factor ELT-3 which can induce AJM-1 and collagen gene expression in non-seam cells, the GATA factors ELT-5 and ELT-6 which redundantly specify seam cell identity (seam cells correspond to lateral epidermal cells, see Fig. 3A). Other potential ELT-1 targets are genes encoding the nuclear hormone receptors NHR-23 and NHR-25, which are essential for embryonic development and control moulting in larvae. The bottom arrows indicate possible targets of these proven or putative transcription factors (see Section 3). for other tissues (Fig. 2) (Newman-Smith and Rothman, act redundantly to activate intestine-specific genes in the 1998; Maduro et al., 2001). According to this model, two embryo, including elt-2 itself, but elt-2 is essential to main- almost identical GATA factors, MED-1 and MED-2, which tain intestinal differentiation (Fukushige et al., 1998; Zhu are direct targets of the maternal gene skn-12, confer et al., 1998; Fukushige et al., 1999). In other words, end-1 mesendoderm identity to a single blastomere (EMS) at and/or end-3 may compensate for the loss of elt-2 in the the 4-cell stage (Maduro et al., 2001). In response to a early embryo, perhaps by up-regulating their own expres- polarising Wnt pathway acting together with a MAP kinase sion. This pathway has probably been evolutionarily pathway, med genes promote endoderm formation by turn- conserved, as GATA factors play an important role in ing on the GATA factors end-1 and end-3, which redun- endoderm specification in other species (Reuter, 1994; dantly specify intestine identity (Zhu et al., 1997; Maduro Reiter et al., 1999), and injection of end-1 mRNA in Xeno- et al., 2001). END-1 in turn activates the expression of pus embryos can trigger the expression of endodermal several transcription factors present in the gut, including specific markers (Shoichet et al., 2000). elt-2, which is another intestine-specific GATA factor The pathway specifying epidermal differentiation reveals (Hawkins and McGhee, 1995). elt-2 and end-1 probably a similar level of redundancy (Fig. 2). As in the intestine, a GATA factor with two zinc-fingers, ELT-1, acts at the top of

2 the hierarchy to specify epidermal identity (Page et al., 1997) In accordance with the general C. elegans nomenclature for genes and and turn on several regulatory genes, including genes encod- proteins, we write gene names in italics with small case letters (i.e. skn-1) and the corresponding proteins with non-italicised capital letters (i.e. ing the GATA factors ELT-3, ELT-5 and ELT-6, and the SKN-1). atypical C2H2 zinc-finger protein LIN-26. Genetic analysis, G. Michaux et al. / Gene 277 (2001) 83–100 87 coupled with ectopic expression experiments, suggests the and maintain the expression of several seam-specific genes, following simplified model for the generation of the ‘major the synthesis of cuticular ridges known as alae and probably epidermal’ cells (Gilleard and McGhee, 2001; Koh and Roth- junction formation. These genes also repress the expression man, 2001; Quintin et al., 2001). In the dorsal and ventral of non-seam genes (elt-3), in particular genes bestowing the epidermis, elt-3, acting redundantly with other genes, turns potential to fuse with other cells (Koh and Rothman, 2001). on and maintains epidermal-specific genes, such as the cuti- In parallel, lin-26 acts to turn on genes that are important for cle collagen gene dpy-7, and the junction marker gene ajm-1 epithelial integrity (‘epithelial-specific’), but is not required (see Section 4) (Page et al., 1997; Gilleard et al., 1999; to turn on genes specifically expressed in the epidermis and Gilleard and McGhee, 2001). In cells of the lateral epidermis not in other epithelia (i.e. cuticle collagens) (Labouesse et al., (seam cells, see Fig. 3A), elt-5 and elt-6 redundantly activate 1994, 1996; Quintin et al., 2001).

Fig. 3. Morphogenesis of epithelial tissues in the embryo. The major epithelial cells are schematised along with their major morphogenetic changes. Anterior is left, dorsal is up. The different organs are not drawn to scale. (A) Epidermal cells are subdivided into ‘major epidermal’ cells, which cover most of the body, and ‘minor epidermal’ cells, which cover other areas and are not represented. Among cells of the ‘major epidermis’, dorsal (top), lateral (middle, dark grey) and ventral (bottom) have different functions and express different regulatory genes. In particular, all major epidermal cells initially express the GATA factor ELT- 1 and the atypical zinc-finger protein LIN-26; subsequently, seam cells express the GATA factors ELT-1, ELT-5 and ELT-6, but not ELT-3 whose expression is restricted to ventral and dorsal cells. The main morphogenetic processes that take place in the embryo are indicated, for further details see recent reviews (Chin-Sang and Chisholm, 2000; Simske and Hardin, 2001). (B) The pharynx has a tube-cyst-tube-cyst structure (see Fig. 1A) (Albertson and Thomson, 1976; Leung et al., 1999). It is surrounded by a basement membrane and is composed of five different cell types. The adult pharynx displays a typical triradiate symmetry in cross-section with a lumen adopting a triangular shape in the buccal cavity, and a Y-shape elsewhere (B-right). Epithelial marginal cells play an important role in giving the lumen its Y-shape: their nuclei migrate towards the basement membrane following cytoplasmic polarisation and their cell bodies retract. (C) The intestine primordium initially displays a radial symmetry except in the first row (C-left). However, soon after intestinal cells become polarised, left ventral (or right ventral) cells intercalate with left dorsal (respectively, right dorsal) cells, resulting in a bilaterally symmetrical tube (C-middle) (Leung et al., 1999). Soon after these cell movements, three pairs of anterior cells move counter-clockwise to the left to initiate a left-handed twist in the anterior part of the intestine; by the time the larva hatches this twist is about 1808 (C-right). 88 G. Michaux et al. / Gene 277 (2001) 83–100 Some of the key genetic data supporting this model are non-seam cells (Wissmann et al., 1997, 1999). The require- mentioned below. Firstly, forced expression of ELT-3 in ment for a gene such as lin-26 was unexpected. It raises the early blastomeres can turn on the expression of AJM-1 and possibility that specific transcription factors control the of the collagen DPY-7, in the same way as forced expres- epithelial cell fate, irrespective of the nature of the epithe- sion of ELT-1 does (Gilleard and McGhee, 2001). lium being made. However, elt-3 inactivation does not affect embryonic The analysis of genes required for epithelial differentia- viability, or epidermal development. This suggests that tion is only starting and remains poorly understood in many other genes act redundantly with elt-3 to maintain epider- cell types. The biochemistry of the above-mentioned mal differentiation, although elt-1 is not a candidate since it proteins is limited, and several conclusions have not yet is down-regulated in dorsal and ventral epidermal cells been confirmed by DNA-binding assays. An intriguing where elt-3 is expressed at mid-embryogenesis (Gilleard question is what confers binding specificity to the different and McGhee, 2001). Secondly, simultaneous inactivation ELT/GATA factors involved in specifying intestinal and of elt-5 and elt-6, which are both expressed in seam cells epidermal cell fates. Many other partially characterised and have related DNA binding domains (80% identical), genes are expressed in the epidermis, in particular several results in embryonic lethality with seam cell defects, or in nuclear hormone receptor homologues. Although their larval lethality with moulting defects. Finally, forced specific targets have not been identified, it is known that expression of LIN-26 in early embryonic blastomeres can their silencing by RNA interference results in lethality turn on expression of AJM-1, DLG-1 and CHE-14 (these (Kostrouchova et al., 1998; Miyabayashi et al., 1999; proteins are involved in junction formation or trafficking), Gissendanner and Sluder, 2000). but not the collagen DPY-7 (Quintin et al., 2001). Although these proteins are normally expressed in lin-26 null mutant embryos their sub-cellular distribution is 4. Epithelial junction structure and assembly affected, suggesting that other genes can compensate for loss of lin-26 function to initiate their expression (Quintin The first major cellular processes that occur in response to et al., 2001). Further evidence that lin-26 is required for genes (such as those mentioned above) that induce differ- epithelial differentiation comes from an analysis of animals entiation of epithelial tissues are the establishment of cell engineered to specifically lack lin-26 expression in their polarity and the assembly of sub-apical junctional belts. somatic gonad. In such animals, AJM-1 is strongly Two major types of junctions can be distinguished by elec- down-regulated in the uterus and the uterine lumen fails tron microscopy in epithelial cells (for general reviews, see to form (den Boer et al., 1998). Drubin and Nelson, 1996; Yeaman et al., 1999). Adherens Two important features of this model should be outlined: junctions (AJs), which are present in vertebrates and inver- the involvement of GATA factors to redundantly control tebrates, are essential for cell adhesion. Tight junctions epidermal differentiation and specify differences among (TJs) in vertebrates and septate junctions (SJs) in Droso- dorsal/lateral/ventral epidermal cells; and the requirement phila are involved in the control of paracellular solute trans- for a specific factor (LIN-26) to turn on genes that are port. These two belts, whose molecular composition is quite important for epithelial integrity. Redundancy in tissue well known in vertebrates and in Drosophila demarcate the specification is a rather general feature in many species, apical from the basolateral domains (Yap et al., 1997; for instance during intestine formation in C. elegans (see Tsukita et al., 1999; Muller, 2000). above), or myogenesis in vertebrates (Arnold and Braun, Electron microscopic examination of C. elegans 2000). The necessity for a differential control among embryonic epithelial cells has revealed only one type of dorsal/lateral/ventral epidermal cells is not surprising, junctional belt in the embryo (Fig. 1D), which has an since these cells perform different morphogenetic func- adherens junction-like structure (Priess and Hirsh, 1986; tions. As outlined in Fig. 3A, dorsal cells intercalate and Leung et al., 1999; Legouis et al., 2000; Koeppen et al., subsequently fuse together, ventral cells mediate the 2001). We will subsequently refer to it as the CeAJ (C. process known as ventral enclosure of the embryo and elegans apical junction). A septate junction has not been seam cells undergo concerted elongation during embryonic observed, except occasionally (D. Hall, pers. comm.; R. elongation. Potential targets of the GATA factors could be Legouis, unpublished data). Below, we discuss junction genes specifically expressed in a subset of epidermal cells formation in the embryo, and particularly in the epidermis. that control morphogenesis of the embryo. For instance, the It is not yet clear whether all junctions share the same C2H2 zinc-finger transcription factor DIE-1, which is components and are assembled in the same way in all C. required for the process of dorsal intercalation, is specifi- elegans epithelial cells. In particular it is unknown if there cally expressed in dorsal epidermal cells (Heid et al., are important differences between the formation of junc- 2001). Similarly, the LET-502 Rho-binding kinase is speci- tions in sheet-like epithelia (i.e. the epidermis) and tube- fically expressed in seam cells and is essential for the like epithelia (i.e. the intestine). concerted elongation process, whereas its antagonist, the Components of the CeAJ have been identified either in a MEL-11 myosin phosphatase, is specifically expressed in screen for monoclonal antibodies recognising membrane- G. Michaux et al. / Gene 277 (2001) 83–100 89 rich or insoluble extracts from embryos (AJM-13), in anchoring remain largely unaffected (Bossinger et al., genetic screens for mutations which affect embryonic 2001; Koeppen et al., 2001; McMahon et al., 2001). Simi- morphogenesis (hmr-1, hmp-1 and hmp-2), or in RNA inter- larly, in ajm-1 mutants the HMR-1/HMP-1/HMP-2 complex ference screens for C. elegans homologues of Drosophila and actin anchoring are unaffected (Koeppen et al., 2001). and vertebrate junction components (dlg-1). AJM-1, the first At the ultrastructural level, inactivation of dlg-1 causes an identified CeAJ component, corresponds to the epitope almost complete disappearance of the electron dense struc- recognised by the monoclonal antibody MH27 (Francis ture of CeAJs where AJM-1 is located, suggesting that and Waterston, 1991). Immunostaining with MH27 reveals DLG-1 functions to aggregate proteins that form this struc- a belt-like pattern around epithelial cells in whole-mount ture (McMahon et al., 2001). Interestingly, AJM-1 and embryos (red in Fig. 1B) (Priess and Hirsh, 1986), and DLG-1 physically interact as shown by yeast two-hybrid immunogold experiments show that AJM-1 localises to and GST-pull down experiments (Koeppen et al., 2001). the electron dense part of the CeAJ at the ultrastructural In ajm-1 mutants, local paracellular gaps of 50–200 nm level (Koeppen et al., 2001). The ajm-1 gene has recently between the normally closely apposed sides of the junc- been identified by expression cloning using MH27 as a tional complex can be observed, showing that AJM-1 is probe, showing that it encodes a large hydrophilic coiled- required to maintain the tightness of the CeAJ electron coil protein with no obvious homology in other species dense domain (Koeppen et al., 2001). The CeAJs of hmr- (Koeppen et al., 2001). The genes hmr-1, hmp-1 and hmp- 1, hmp-1 and hmp-2 mutants have not yet been examined by 2 encode E-cadherin, a-catenin and b-catenin homologues, electron microscopy; it is not clear whether one should respectively (Costa et al., 1998). HMP-2 is one of three C. expect them to be normal since HMP-1 is more apical elegans b-catenin homologues. It is the only b-catenin than AJM-1, or defective. Surprisingly, neither a loss of homologue to interact with HMR-1 (the closest E-cadherin the HMR-1/HMP-1/HMP-2 complex, nor of the DLG-1/ homologue in the C. elegans genome), but it has lost a AJM-1 complex, strongly affects cell adhesion and polarity function associated with classical b-catenins, the potential (Costa et al., 1998; Raich et al., 1999; Bossinger et al., 2001; to signal in a Wnt pathway. Instead, Wnt signalling involves Koeppen et al., 2001; McMahon et al., 2001). A reasonable BAR-1, another b-catenin homologue, or WRM-1 which hypothesis is that both complexes act redundantly to main- acts in a non-conventional manner (Rocheleau et al., tain adhesion and cell polarity. In addition, DLG-1 might be 1997; Eisenmann et al., 1998; Korswagen et al., 2000). required to maintain some unique aspects of cell polarity, Staining with antibodies against HMP-1 and HMR-1 reveal since the apical protein CRB-1 (see below) is no longer in first approximation a belt similar in position and structure detected in the intestine of embryos lacking DLG-1 to that formed by AJM-1 (Costa et al., 1998). The most (Bossinger et al., 2001). recently identified CeAJ component, named DLG-1, is the The results summarised above suggest that the CeAJ C. elegans orthologue of Drosophila Lethal Discs Large combines the functions of both adherens junctions and (Dlg) and human hDlg proteins and colocalises with AJM- septate/tight junctions in other species. Thus, although C. 1 in epithelial cells (Bossinger et al., 2001; Koeppen et al., elegans epithelial cells do not contain a distinguishable 2001; McMahon et al., 2001). septate junction, the CeAJ appears to be more similar to A combination of genetic, immunochemical and electron epithelial junctions in other species than initially antici- microscopic studies suggest that the apical junction of C. pated. Interestingly, in vertebrates, adherens and tight junc- elegans epithelial cells contain two independent units with tions might also be two aspects of a single unit, since the at least partially distinct functions (Fig. 1D). The first and tight junction protein ZO-2 can also associate with adherens most apical unit would comprise HMR-1, HMP-1, HMP-2 junctions (Itoh et al., 1999). In addition, recent results and possibly other proteins, while the other unit would suggest that a second complex, the afadin/nectin complex, include at least AJM-1 and DLG-1 (Koeppen et al., 2001; contributes to cell adhesion in addition to the cadherin/cate- McMahon et al., 2001). Indeed, as observed by immunos- nin complex (Tachibana et al., 2000). Some unique proper- taining, AJM-1 and DLG-1 do not appear to colocalise with ties of the CeAJ, in particular the likely redundancy in the HMP-1 in transverse sections through the apico-basal axis maintenance of adhesion and polarity, make C. elegans a of epidermal cells (McMahon et al., 2001) or in the pharynx particularly attractive model to dissect the functions of junc- (Koeppen et al., 2001). Mutations in hmr-1, hmp-1 and hmp- tional proteins, such as actin anchoring by HMP-1 or protein 2 do not affect the distribution of AJM-1, but cause defects aggregation by DLG-1. Indeed, in Drosophila and mammals in actin cytoskeleton organisation and anchoring (Costa et the severe polarity defects associated with loss of cadherin/ al., 1998; Raich et al., 1999). Conversely, in the absence of catenin or Dlg function make it more difficult to study these DLG-1, AJM-1 distribution becomes very irregular, functions in a live embryo. whereas the HMR-1/HMP-1/HMP-2 complex and actin How junctions become positioned sub-apically has been revealed by characterisation of the gene let-413 (Legouis et al., 2000). This gene was identified through a screen in 3 AJM-1 was originally called JAM-1 (Mohler et al., 1998; Raich et al., 1999) but has recently been renamed to avoid any possible confusion with which embryos homozygous for chromosomal deficiencies the vertebrate tight junction protein JAM-1 (Koeppen et al., 2001). were examined for their AJM-1 pattern (Labouesse, 1997). 90 G. Michaux et al. / Gene 277 (2001) 83–100 In let-413 mutants, the AJM-1 distribution within epithelial 5. Hemidesmosomes, intermediate filaments and the cells is severely disrupted. LET-413 is a basolateral plasma connection with other tissues membrane protein, which possesses one PDZ domain and sixteen leucine-rich repeats (LRR domain). The LRR Epithelial cells are also tightly linked to non-epithelial domain is quite similar to the Ras-binding protein SUR-8, cells through the extracellular matrix. These interactions, raising the possibility that LET-413 could bind a GTPase. which must be integrated throughout development, are Electron microscopic and confocal analysis revealed that in essential to ensure mechanical coupling between adjacent let-413 mutants CeAJs and their components are spread tissues, to maintain tissue architecture and to facilitate cell- along the lateral membrane (Legouis et al., 2000; Koeppen cell communication (for review, see Hynes, 1999). et al., 2001; McMahon et al., 2001). Interestingly, the distri- Several studies have recently highlighted the role of bution of junction components in let-413 deficient embryos hemidesmosomes and intermediate filaments (IFs) in the resembles that observed in immature wild-type epidermal epidermis to link the underlying muscle on its basal surface cells, where markers are initially located along the lateral and the external cuticle on its apical surface (Fig. 1E). In C. membrane, instead of sub-apically (McMahon et al., 2001). elegans as in many invertebrates, the cuticle acts as an These observations suggest that in epidermal cells let-413 exoskeleton onto which muscle cells attach to allow loco- acts in a process leading to the coalescence of junction motion. Sarcomeres are mechanically coupled to the basal components at a sub-apical position, or possibly to prevent lamina that separates muscles from the epidermis through junction assembly laterally. The function played by LET- integrins (Hresko et al., 1994; Williams and Waterston, 413 is likely to be general since the Drosophila homologue 1994). In regions of muscle contacts, epidermal cells Scribble has a directly related role and subcellular localisa- become flattened and contain IF arrays connecting electron tion (Bilder et al., 2000a,b). In vertebrates, ERBIN, one of dense structures similar to hemidesmosomes, which the human LET-413 homologues, is a basolateral protein together form what is known as fibrous organelles (Francis required to localise the EGF receptor ERBB2/HER2 at the and Waterston, 1991). IFs represent a major cytoskeletal basolateral membrane (Borg et al., 2000). system found in animal cells that transmit mechanical stress In Drosophila, two additional complexes, localised apical (Fuchs and Cleveland, 1998). The diversity of this family to the AJ, are essential to establish cell polarity and assemble (more than 50 genes in mouse or human) explains the poor AJs. One complex involves aPKC, an atypical protein kinase understanding of its functions. In C. elegans, there are only C, the PDZ proteins Bazooka and Par-6 (Kuchinke et al., 11 genes coding for IFs, which can be divided into five 1998; Schober et al., 1999; Wodarz et al., 2000; Petronczki groups A1–A4, B1–B2, C1–C2, D1–D2, E1. Inactivation and Knoblich, 2001), and the other includes the transmem- by RNA interference has revealed that four of them (A1– brane protein Crumbs with the multi-PDZ protein Disc Lost A3, B1) are essential for embryonic or larval development (Bhat et al., 1999; Klebes and Knust, 2000). Bazooka, Par-6 (Karabinos et al., 2001). In embryos and larvae lacking the and aPKC are the respective homologues of the worm A2 and A3 IFs, muscle cells become detached from the proteins PAR-3, PAR-6 and PKC-3, which colocalise at the body wall although it is not yet known at which level this anterior cortex of the early C. elegans embryo where they are detachment takes place. Consistent with a role in epidermal essential to establish embryonic polarity (for reviews, see cells, GFP fusion experiments indicate that A3, and possibly Rose and Kemphues, 1998; Doe and Bowerman, 2001). A2, are specifically expressed in the epidermis (Karabinos Their vertebrate homologues ASIP, mPAR6 and PKCz are et al., 2001). This confirms the essential role of IFs in epithe- apical in mammalian epithelial cells, and also form a lial cells to establish and maintain connections between complex together with the GTPase CDC42 (for a review, several tissues during organogenesis. see Kim et al., 2000). In C. elegans, PAR-3, PAR-6 and Two screens for mutants presenting detachment of muscle PKC-3 are also present at the apical surface of intestinal cells have led to the identification of the genes mua-3 (fragile and pharyngeal cells (green in Fig. 1B) but their role in muscle attachment) and mup-4 (muscle positioning defec- these cells has not yet been examined (Leung et al., 1999). tive) (Gatewood and Bucher, 1997; Plenefisch et al., 2000), There is also a Crumbs homologue in C. elegans, CRB-1, which have recently been cloned (Bercher et al., 2001; Hong which is localised at the apical surface of intestinal cells. et al., 2001). In these mutants, muscle cells are detached from However, RNA interference against crb-1 or against a disc the body wall and animals die during embryonic or larval lost homologue does not affect cell polarity or junction stages. Electron microscopy analysis has shown that the assembly (Bossinger et al., 2001). epidermis detaches from the cuticle and is less strongly flat- In summary, there are some differences in the specific tened in these mutants, but that muscles remain attached to functions that homologous proteins play in epithelial cells the epidermis (Bercher et al., 2001; Hong et al., 2001). MUA- from C. elegans and other systems (Drosophila and verte- 3 and MUP-4 are single pass transmembrane proteins with a brates). Nonetheless there are also common features, one of large extracellular domain mainly formed of EGF repeats and which is the presence of homologous proteins at the same a cytoplasmic domain with weak homology to filaggrins, positions. Future studies and comparisons will certainly which are known to bind intermediate filaments (Steinert et reveal the relevance of these similarities and differences. al., 1981). Amino acid identity between MUA-3 and MUP-4 G. Michaux et al. / Gene 277 (2001) 83–100 91 reaches 50% in the extracellular region. Both proteins are Genetic analysis of somatic gonad morphogenesis has expressed in the epidermis and localised at sites of muscle identified a new basal lamina component, GON-1, which contacts, in a pattern similar to that of intermediate filaments. is a secreted protein with a metalloprotease domain related The authors suggest that MUA-3 and MUP-4 act at the to the ADAM family and thrombospondin repeats. GON-1 epidermal apical surface, binding on the one hand to the is required for two aspects of gonad morphogenesis (Blel- cuticle via their extracellular domain, and on the other to loch et al., 1999; Blelloch and Kimble, 1999). In gon-1 intermediate filaments presumably through their filaggrin- mutants, the somatic gonad primordium and uterus fail to like cytoplasmic tail (Bercher et al., 2001; Hong et al., 2001). acquire a clear epithelial character, in particular the uterine Another transmembrane epidermal protein required for lumen is essentially absent, suggesting that GON-1 muscle attachment is called myotactin. The gene encoding promotes their epithelialisation. It might do so by providing this protein, let-805, was isolated by screening an expres- a basal clue important to polarise somatic gonadal cells. sion library with the monoclonal antibody MH46 (Hresko et Interestingly, mutations eliminating the a-laminin chain al., 1999), which was identified in the same screen as MH27 LAM-3 affect organogenesis of the pharynx, and in parti- (see Section 4) (Francis and Waterston, 1991). The extra- cular the position at which some CeAJs are assembled (W. cellular region of myotactin contains 32 fibronectin type III Wadsworth, pers. comm.). These observations bring genetic (FNIII) repeats, but its cytoplasmic part does not present any support to the current models regarding the influence of homology. Immunohistochemical analysis suggests that basal cues on the establishment of cell polarity, although myotactin is localised at the basal surface of epidermal its influence may be subtler than anticipated. Independently cells. Based on its number of FNIII repeats, it could expand of its function in setting polarity, GON-1 is also essential for through the basal lamina to allow anchorage of muscle and the initial migration of the leader cells (distal tip cells, or maintain the association with the fibrous organelles. In let- DTC, in hermaphrodites; linker cell, or LC, in males) which 805 mutants, muscles detach when contraction begins at the guide growth and extension of the somatic gonad. MIG-17, two-fold stage, and fibrous organelles tend to be localised another metalloprotease related to GON-1 but lacking the throughout the epidermis instead of just in areas of muscle thrombospondin repeats, is required for a distinct phase of contact. However fibrous organelles remain surprisingly DTC/LC migration. It is likely that the function of GON-1 well organised and maintain a regular pattern, suggesting and MIG-17 in cell migration is to locally degrade the basal that myotactin may only be required to maintain the asso- lamina to allow further migration or to reorient the direction ciation between muscle and fibrous organelles. of migration (Blelloch et al., 1999; Blelloch and Kimble, The analysis of the muscle-epidermis-cuticle connection 1999; Nishiwaki et al., 2000). is providing exciting results at a rapid pace, identifying new Hemicentin, which is secreted by muscle and gonadal proteins with known modules. Genetic analysis in the worm leader cells, is another novel extracellular matrix protein embryo should complement biochemical studies in verte- identified by genetic analysis (Vogel and Hedgecock, brates to better understand the complex and still poorly 2001). Hemicentin, which is encoded by the gene him-4, understood process of mechanical coupling between tissues. contains 48 immunoglobulin repeats flanked by new terminal domains. The HIM-4 protein forms fine tracks at specific sites, particularly in regions of oriented linear junctions, 6. Basement membrane and organogenesis which can always be correlated with phenotypes observed in him-4 mutant larvae. These mutants present pleiotropic Epithelial cells are characterised by the presence of a defects in different organs, such as defective gonadal move- basal lamina, which provides a link with the underlying ment, loss of adhesion of the intestine to body wall muscles, tissue and an important polarity clue (for a review, see detachment of the uterus from the lateral epidermis once Yeaman et al., 1999). For instance, experiments with tissue egg-laying begins (see Section 10 and Fig. 4). It has been culture cells have shown that the presence of a basal lamina suggested that hemicentin organises hemidesmosomes in can induce polarisation of MDCK cells (Wang et al., the overlying epidermis where the uterus contacts the lateral 1990a,b). Because of its anatomical and genetic simplicity epidermis, and along mechanosensory axons. C. elegans, which has many classical components of the extra-cellular matrix such as laminins, nidogen, perlecan, type IV collagen (Kramer, 1997), is a good model to identify 7. Differential trafficking to apical and basolateral new components of the basement membrane or to clarify the membrane domains roles of proteins already known in vertebrates. For instance, C. elegans has only two a-laminin chains (LAM-3 and EPI- An important aspect of epithelial cell polarity concerns 1) instead of five in vertebrates, only one b-integrin chain the differential trafficking of proteins to the apical and baso- (PAT-3) compared to eight in vertebrates and only two a- lateral domains. Proteins destined to the apical and basolat- integrin chains (PAT-2 and INA-1) instead of 17 in verte- eral membranes are often sorted out in the Golgi based on brates (Hutter et al., 2000). Below, we will focus mainly on specific determinants (Mellman and Warren, 2000). Di- components that affect epithelial cells. leucine and tyrosine residues can mediate basolateral target- 92 G. Michaux et al. / Gene 277 (2001) 83–100 ing, whereas O-glycosylation, N-glycosylation or a GPI membrane (Ikonen and Simons, 1998). Biochemical analy- adduct (glycophosphoinositol) can mediate apical targeting. sis has led to the identification of several raft components, The mechanisms that control protein trafficking in C. including caveolin. RNA interference studies indicate that elegans are just beginning to be analyzed. caveolin may not be essential for apical trafficking in C. In mammalian MDCK cells, Simons and Ikonen have elegans (Scheel et al., 1999). Genetic analysis has led to proposed that lipid microdomains enriched in cholesterol identification of che-14, which encodes a probable compo- (also called lipid rafts) direct trafficking towards the apical nent of the apical trafficking machinery in ectodermal G. Michaux et al. / Gene 277 (2001) 83–100 93 epithelial cells. Ultrastructural studies showed that che-14 the basolateral membrane of VPCs. Its basolateral restric- mutants accumulate vesicles close to the apical membrane tion is achieved by a complex of three PDZ-containing and have a marked reduction of the cuticle thickness (i.e. an proteins encoded by the genes lin-2, lin-7 and lin-10 apically secreted structure), causing larval lethality or (Hoskins et al., 1996; Simske et al., 1996; Kaech et al., abnormal behaviour (Michaux et al., 2000). Since the 1998; Whitfield et al., 1999). Mutations in these genes result CHE-14 protein itself is predominantly located at the apical in a vulvaless phenotype due to apical mislocalisation of surface of ectodermal epithelial cells, it has been suggested LET-23. Elegant experiments have shown that the type I that it is necessary for normal trafficking of a subset of PDZ domain of LIN-7 interacts with the final residues of proteins to the apical domain (Michaux et al., 2000). Inter- LET-23, which correspond to a canonical type I PDZ target estingly, CHE-14 is a member of the NPC1/Patched/ sequence. LIN-7 can also directly interact with LIN-2, Dispatched family of multipass transmembrane proteins, which can bind LIN-10 (Kaech et al., 1998). The LIN-7 which contain a putative sterol-sensing domain (Lange protein appears to colocalise with junctions, while LIN-10 and Steck, 1998). Consistent with CHE-14 trafficking func- is a presumptive Golgi protein (Simske et al., 1996; Whit- tion, the vertebrate NPC1 protein is thought to control field et al., 1999), indicating that LIN-2/LIN-7/LIN-10 may cholesterol trafficking between late endosomes and lyso- not act at the same step in the trafficking pathway that somes (Neufeld et al., 1999; Cruz et al., 2000), while the prevents LET-23 from going to the apical membrane. This phenotype of flies mutated in dispatched may result from pathway has been conserved in many species and also oper- defective secretion of the morphogen Hedgehog (Burke et ates in non-epithelial cell types, particularly in the nervous al., 1999). However, the targets of CHE-14 and the precise system (Bredt, 1998). Analysis of LET-23 localisation in C. step in exocytosis to the apical membrane at which CHE-14 elegans vulval precursors allows a powerful genetic dissec- acts remain to be identified. It should be pointed out that loss tion of this pathway. of CHE-14 activity affects epidermal cells in a different way than mutations in LRP-1, a gp330/megalin-related protein necessary for cholesterol endocytosis, which result in an 8. Morphogenesis of the intestine inability to shed and degrade the old cuticle at each larval moult (Yochem et al., 1999). The C. elegans intestine is an extraordinarily simple bilat- The mechanism involved in targeting one particular baso- erally symmetrical organ containing only 20 cells that lateral protein, the EGF receptor homologue LET-23, has derive from a unique blastomere, the E blastomere. Most been particularly well characterised. LET-23 is involved in of the intestine is a tube composed of rings of only two cells, several signalling events, particularly during vulva forma- such that the lumen of the intestine is a canal between two tion (see Figs. 4A and 5). Normally, LET-23 receives a opposed cells (Figs. 1C and 3C). Elegant work from the signal made by the anchor cell (AC), which is located dorsal Priess laboratory recently described some of the cellular to the basolateral surface of vulval precursor cells (VPCs). and genetic steps involved in intestinal morphogenesis Thus, to receive the AC signal, LET-23 should be present at (Leung et al., 1999; Hermann et al., 2000). It involves cyto-

Fig. 4. Morphogenesis of the egg-laying system. (A) The 12 cells that constitute the ventral epidermis in the embryo (see Fig. 3A) divide in the first larval stage to each generate an epidermal cell (Pn.p) and a neuroblast (Pn.a). The fates of Pn.p cells are symbolised in the top right drawing. At the first larval stage, six Pn.ps fuse with the major epidermal syncytium hyp7 (F fate), while the others become vulval precursor cells (VPCs). At the third larval stage, the three central cells (P(5-7).p) are induced by the anchor cell (AC in blue) to generate the vulva and adopt either a 18 or 28 fate, while the three other Pn.ps adopt the 38 fate (one division and fusion of both daughters to hyp7). The 18 and 28 fates are characterised by three cell divisions along specific cleavage planes to generate eight and seven cells, respectively, with distinct properties The 22 vulval cells generated by P(5-7).p can adopt seven different fates following a bilateral symmetry, from the outermost vulA to the innermost vulF rings (top left drawing). Each fate is executed either by two or four cells, which undergo short-range migrations and extension of their apical domains to wrap around the more proximal cells and to fuse with other cells of the same fate. Apical extension and fusion push internal rings dorsally and thereby drive the autonomous invagination process. Each ring has a specialised function: vulA connects the vulva to the surrounding epidermis syncytium hyp7; vulE anchors the vulva to the lateral seam cells; vulF connects the vulva to the uv1 uterine cells; vulD and vulF anchor the sex muscles that open the vulva. As explained further in Section 10, the AC by fusing with the utse plays a critical role in making the connection between the vulva and uterine lumens. (B) The uterus is composed of 61 cells that derive from three ventral (VU) and two dorsal (DU) uterine precursors (top left drawing). Each VU descendant generates four intermediate precursors, which adopt a so-called p or r fate depending on whether they are close to the AC or not. After cell fusion, these 61 cells generate on each side of the vulva (from distal to proximal) one spermatheca-uterine valve (sujn), four uterine toroids (ut) and three uterine-vulval cells (uv1-3), and centrally a dorsal uterine cell (du) and an H-shaped uterine-seam cell (utse) (top left and top right drawings). Formation of the uterine lumen may be related to formation of the intestinal lumen. The four ut and the sujn cells on each side of the vulva are formed by the fusion of four to six cells generated by DU and r descendants. In the central part of the uterus, the ventral uv2-3 cells are mononucleate, the dorsal du cell results from the fusion of four cells, and the H-shaped utse results from heterotypic fusion of 8 p descendants with the AC. Each uterine cell has a specialised function: uv2 sends cytoplasmic processes contacting the dorsal uterus; uv1 physically connects the uterus to the vulva by forming apical junctions with the utse and the vulF cells of the vulva; the utse anchors the uterus to the lateral epidermis. The top right drawing show a longitudinal-section through the uterus, the bottom right drawing shows a transverse-section through the uterus and the vulva. Note the specialised projections made by vulE, uv1, uv2, utse cells, and the positions of vulva opening muscles. Insets correspond to the vulval primordium (A) or the entire egg-laying system (B; sujn cell, arrow; ut cells, diamonds) of transgenic animals expressing a dlg-1::gfp translational fusion, highlighting junctions at the same stage as the drawing above (A) or to the right (B). Drawings in this figure are adapted from Newman and Sternberg (1996) and Sharma-Kishore et al. (1999). Scale bar 10 mm (A) or 40 mm (B). 94 G. Michaux et al. / Gene 277 (2001) 83–100

Fig. 5. Signalling cells involved in generating the egg-laying system. The cartoon shows the known signalling and receiving cells involved in vulva and uterus formation (see also Fig. 4). Several parallel pathways determine patterns of cell division and differentiation, and organogenesis of the egg-laying system. The nature of the signalling outputs is symbolised by different arrows (key on the right). The following signalling pathways have been involved in patterning the egg-laying system (Greenwald, 1998; Sternberg and Han, 1998; Moghal and Sternberg, 1999; Fay and Han, 2000). The SynMuv pathway, which acts through a retinoblastoma (Rb) homologue, promotes fusion of VPCs to the main hypodermal syncytium hyp7, but is overridden by AC signalling. The EGF/Ras /MAP kinase pathway mediates induction from the AC to VPCs or from vulF to uv1 (fusion of p descendants to form the utse appears to be a default fate). The LIN- 12/Notch pathway mediates lateral signalling between 18 and 28 VPC precursors and induction of uterine p precursors by the AC. An APC/b catenin pathway, which acts on the Hox gene lin-39, is essential for expression of the vulval cell fate. The heterochronic gene lin-29 coordinates the timing of development. The presumptive transcription factors LIN-11 and COG-2 are essential to build the vulval-uterine connection. plasmic polarisation of each cell, microtubule-dependent left-right asymmetry within the intestinal tube and subse- nuclear migration to the midline, intercalation of ventral quently allow the anterior pairs of cells II–IV to rotate. Ante- cells with dorsal cells in a stereotyped pattern that does rior-posterior and left-right asymmetries within the intestine not cross the left/right boundary, formation of the lumen, are specified by a MAP kinase-like and a Notch-like pathway counter-clockwise rotation of three anterior pairs of cells that are repeatedly used in the early embryo (Hermann et al., and appearance of a basement membrane (Leung et al., 2000). These pathways might modify the adhesive properties 1999). The separation of apical membranes at the midline, of left versus right cells in the anterior part of the intestine and which initiates lumen formation, coincides with the appear- create favourable conditions to allow a rotation. The anterior- ance of vesicles at the apical surface (Leung et al., 1999). posterior asymmetry is generated within the intestinal These vesicles resemble some endocytic vesicles, raising primordium at or after the third division of the E blastomere, the possibility that endocytosis might contribute to cell which occurs along the anterior-posterior axis. It involves the separation. Cytoplasmic polarisation and formation of a LIT-1 kinase, the final effector of a non-conventional MAP lumen are cell-autonomous processes that occur in embryos kinase pathway that results in excluding the Tcf/Lef homo- containing only descendants of the E blastomere. However, logue POP-1 from posterior daughters after anterior-poster- intercalation does not occur in these partial embryos, such ior divisions (Kaletta et al., 1997; Meneghini et al., 1999; that the intestinal cyst made retains a radial symmetry Rocheleau et al., 1999). In parallel, a Delta-related ligand instead of acquiring a bilateral symmetry (Leung et al., encoded by the gene lag-2 down-regulates expression of 1999). It suggests that external clues play a role in the the Notch-related receptor LIN-12 in the left part of the intes- acquisition of a bilateral symmetry. tine primordium (for a review of the LIN-12/Notch pathway, The genetic control of these events is not understood, see Greenwald, 1998). All intestinal cells initially express except for the events that induce an anterior-posterior and a LIN-12, while only cells located on the right side of the intes- G. Michaux et al. / Gene 277 (2001) 83–100 95 tine express LAG-2. The initial LIN-12 asymmetry serves to 10. Patterning and morphogenesis of the egg-laying pattern a second, subsequent, LIN-12 mediated interaction. system In lin-12, lag-2,orlag-1 (which encodes a Su(H) homologue) mutants, anterior cells fail to rotate (Hermann et al., 2000). It The egg-laying system of C. elegans comprises an exter- is likely that the basic mechanisms C. elegans uses to estab- nal epithelial tube, the vulva, an internal epithelial tube, the lish polarity in its 20-cell intestine will be relevant to studies uterus, sex muscles and neurons. Sex muscles act to open on organogenesis in more complex systems. the vulva or squeeze the uterus. Historically, formation of the egg-laying system was the first to be studied by genetic analysis, and as a consequence is the best known (Horvitz 9. Morphogenesis of the pharynx and Sulston, 1980). Although its development is compara- tively more complex than any other organ discussed so far, The pharynx, which corresponds to the C. elegans fore- it remains much simpler than most vertebrate organs, and gut, is more complex than other embryonic organs as the offers a unique system in which to dissect the signalling mature organ includes glands, neurons, epithelial and myoe- pathways involved in patterning and morphogenesis of pithelial cells. Most cells are polarised and their apical epithelia. Formation of the egg-laying system involves the surfaces border a Y-shaped, cuticle-lined lumen. They are specification of different specialised epithelial cells, fusion generally arranged in sets of nine cells with three-fold rota- of adjacent specialised cells, and finally connection of the tional symmetry around the lumen (Albertson and Thom- uterus and vulva. The main cellular consequences of signal- son, 1976). Two recent studies have investigated some of ling events that pattern vulval and uterine cell fates are to the events that occur during formation of the pharyngeal induce specific short-range cell migrations and fusions. A lumen and the buccal cavity (Leung et al., 1999; Portereiko detailed discussion of all these pathways is beyond the and Mango, 2001). Cytoplasmic polarisation appears later scope of this article (for earlier reviews, see Newman and in the pharyngeal primordium than in the intestinal primor- Sternberg, 1996; Sternberg and Han, 1998; Delattre and dium, when cells become wedge-shaped, with their narrow Felix, 1999; Moghal and Sternberg, 1999; Fay and Han, apical surface extending to the midline and their nuclei 2000; Shemer and Podbilewicz, 2000). Instead, we provide moving to the basal surface (Leung et al., 1999). As in the an overview of vulval and uterine development and focus on intestine, formation of the lumen starts by a separation of some key events related to the connection of both organs. adjacent membranes at the apical midline. Subsequently, and unlike what happens in the intestine, three epithelial 10.1. The vulva, a stack of concentric rings cells called the marginal cells retract their apical tips, thereby considerably expanding the apical surface and The vulva is built from 22 epithelial cells which, after giving rise to the Y-shaped lumen characteristic of the phar- their fusion by groups of two or four cells sharing the same ynx (Fig. 3B) (Leung et al., 1999). The mechanisms fates (homotypic fusion), form a stack of seven concentric involved in marginal cell retraction are unknown. rings. These 22 cells originate from three ventral epidermal Portereiko and Mango (2001) examined the cellular events precursors, which are progressively selected in two steps involved in connecting the pharynx primordium to the buccal among 12 cells called the Pn.p cells (Fig. 4A). In the first cavity. They have shown that it involves three main steps. step, during the first larval stage, the Deformed-like Hox First, the apical/basal axis (as defined by the position of gene lin-39 confers the potential to become vulval precursor CeAJs) of the anterior-most epithelial cells within the cells (VPCs) to the central epidermal cells (P(3-8).p) (Clark primordium, which is initially oriented along the anterior- et al., 1993; Salser et al., 1993; Ch’ng and Kenyon, 1999). In posterior axis towards the centre of the primordium, rotates the second step, during the third larval stage, the anchor cell approximately 908 to orient along the dorsal ventral axis. (AC) located in the overlaying somatic gonad induces P(5- Meanwhile, the basement membrane apposed to the anterior 7).p to form the vulva (Kimble, 1981). The AC signal, an surface of these cells disappears, which transforms the cyst- EGF-like signal which is transduced by the Ras/MAP kinase like pharyngeal primordium into an open tube. Second, the pathway also initiates vulval patterning. Normally P6.p so-called arcade cells, which are anterior to the pharynx adopts a fate called 18, which is to generate the two inner- primordium and will form the buccal cavity, become epithe- most vulval rings that are closest to the uterus, while P5.p lial and produce a continuous epithelium linked to the epider- and P7.p adopt a fate called 28, which results in the genera- mis anteriorly and to the developing pharynx posteriorly. tion of the outermost vulval rings (Sternberg and Horvitz, Third, the apical surfaces of arcade cells constrict, thereby 1986). Genetic analysis and cell ablation experiments have pulling the entire pharynx anteriorly. Surprisingly, none of shown that the graded nature of the AC signal, in conjunc- the genes that have been mentioned so far affect pharynx tion with lateral signalling between P(5-7).p cells mediated organogenesis, except let-413 (Legouis et al., 2000), epi-1 by the LIN-12/Notch receptor, ensures that only the cell and lam-3 (W. Wadsworth, pers. comm.). Genetic analysis of closest to the AC adopt the 18 fate (Figs. 4A and 5) (Stern- pharynx formation should identify genes playing an impor- berg and Han, 1998; Moghal and Sternberg, 1999). Both tant role in forming tubes or connecting cysts. pathways interact through a MAP kinase phosphatase, 96 G. Michaux et al. / Gene 277 (2001) 83–100 called LIP-1, which in response to LIN-12 signalling down- 10.3. Making the connection and co-ordinating vulva and regulates Ras/MAP kinase signalling in cells adopting the 28 uterus patterning fate (Berset et al., 2001). Each 18 or 28 precursor divides three times in a specific manner to generate vulval cells that The LIN-12 signalling pathway and its effectors (Green- fuse to form the seven rings with specific functions, called wald, 1998), play a key role in making the vulva-uterus vulA to vulF (Fig. 4A). The AC again plays an important connection. It specifies the p fate upon AC induction and role, together with the Ras and a Wnt pathways, to pattern thus allows the generation of the centrally located uterine the 18 lineage (Wang and Sternberg, 2000). The process of cells that make the connection (utse and uv1 cells) cell fusion and ring formation transforms the linear array of (Newman et al., 1995). One of the final LIN-12 targets in cells present in the vulva primordium into a sequence of p cells is the LIM-homeodomain protein LIN-11 (Newman toroidal syncytia (Sharma-Kishore et al., 1999; Shemer et et al., 1999). In lin-11 mutants, the p descendants that al., 2000). contribute to the utse are mis-specified and fail to fuse with the AC, resulting in the absence of a connection (Newman et al., 1999). Formation of the utse requires the 10.2. The uterus, a set of toroids linked to the vulva by activity of a second transcription factor, COG-2, a member specialised cells of the SOX family which is expressed in p descendants. In cog-2 mutants, the p fate is normally specified but the AC The uterus is initially composed of 61 cells, which fuse to fails to fuse with the utse (Hanna-Rose and Han, 1999), form seven different cell types (Fig. 4B): distal to the vulva suggesting that COG-2 acts at a late step, possibly by and on each side of it, it consists of a series of four toroids; directly controlling the fusion process. centrally, it consists of three pairs of cells that link the vulva Making the connection between the vulva and the uterus, to the toroids, and of a fourth large H-shaped cell that allows and anchoring both organs to the lateral or surrounding the connection to be made. Uterine cells derive from three epidermis, requires that vulval, uterine and other epidermal ventral (VU) and two dorsal (DU) uterine precursors. The cells reach the appropriate developmental stage at the same AC plays three important roles in patterning the uterus. time. Therefore, there must be genes that act to keep vulval, During the third larval stage, it induces six of the VU grand- uterine and surrounding epidermis development in register. daughters that are closest to it to adopt the uterine p fate, One such gene is lin-29, which encodes a zinc-finger protein while the six more distal granddaughters adopt the default r and is a member of the heterochronic gene family (Bettinger fate (Figs. 4B and 5). Normally, r descendants contribute to et al., 1997; Newman et al., 2000). Mutations in heterochro- form the distal toroids and the uterine-vulval cells uv2-3, nic genes cause reiterated or precocious programs of devel- while p descendants contribute to form the centrally located opment. In lin-29 mutants, there is no terminal differentiation cells (i.e. uv1 cells and the uterine-seam cell or utse). Speci- of the lateral epidermal cells, abnormal specification of p fication of the uv1 fate involves a reciprocal EGF signal cells which all fuse to form the utse, no vulval-uterine from vulF to induce uv1 differentiation among a subset of connection and abnormal eversion of the vulva. LIN-29 is p descendants (Chang et al., 1999). The utse also plays an expressed in the AC where it acts upstream of LAG-2, the important function in connecting the uterine and vulval LIN-12 ligand (Greenwald, 1998), in the VPCs and their lumens. At the fourth larval stage, the AC partially pene- descendants, in p cells where it acts upstream of LIN-12, trates the basal lamina that separates the growing uterus and in lateral epidermal cells. Therefore lin-29 acts to co- from the innermost vulval ring (vulF) and contacts both ordinate development of all three tissues. tissues (the mechanisms of this invasion are not known). Continuous analysis of vulval and uterine development Subsequently, the AC fuses with the utse, and its nucleus should reveal how signalling can induce cell shape changes undergoes a migration towards the lateral extension of the and identify possible targets of the numerous transcription utse. This leaves only a thin lamellar process above the factors involved. Some key issues that should be addressed vulva which can be broken by the first egg laid (Fig. in the future include the determination of how vulval rings 4A,B). The final connection between the vulva and the or uterine toroids arise. What are the mechanisms involved uterus requires that CeAJs physically link the innermost in short-range migrations that precede cell fusion and are ring vulF to the central-most ventral uterine cells (uv1). they related to the process of ventral enclosure in the Patterning of the vulva and the uterus thus involves three embryo? What drives homotypic or heterotypic cell fusion general principles: cell fate potentials are restricted in a and are specific homophilic adhesion molecules involved? stepwise manner; one cell, the AC, has many properties of One strategy will come from a genetic analysis of mutants an ‘organiser’; and reciprocal signalling from the vulva back specifically affecting late aspects of vulva morphogenesis to the uterus ensures a linkage of both organs. The AC can and invagination, resulting in a protruding (Eisenmann and be considered as an organiser, because it induces different Kim, 2000) or a collapsed vulva (Herman et al., 1999). For fates within the vulva and the uterus, makes the connection instance, three of the genes defined by mutations resulting in between both tubes possible by invading the basal lamina, a collapsed squashed vulva (sqv-1-8 genes) have been and finally fuses with a larger cell to clear the way for eggs. cloned and belong to the glycosylation pathway; they may G. Michaux et al. / Gene 277 (2001) 83–100 97 modify cell-cell or cell-basal lamina interactions (Herman analysis (yeast) and more complex multicellular organisms and Horvitz, 1999). In parallel, ongoing studies of vulval where genetics is difficult and/or costly (vertebrates) but development in the nematodes Pristionchus pacificus and biochemical analysis is easy. A comparison between related Oscheius (Eizinger and Sommer, 1997; Dichtel et al., 2001) processes in vertebrates, Drosophila, yeast as well as other may prompt a reinvestigation of old problems and lead to nematodes should give clues about conserved functions. the discovery of non-suspected functions. A recent example comes from ablation studies performed a few years ago in the nematode Oscheius, which had shown that induction of Acknowledgements the vulva requires two nested inductions from the gonad (Felix and Sternberg, 1997). More recently, some careful We are grateful to Marie-Anne Fe´lix and Laura McMa- analysis of the 18 fate with new probes, showed that in C. hon for critical reading of the manuscript and useful discus- elegans too the AC is necessary not only to induce vulva sions. We thank Jeff Hardin, John Gilleard, Susan Mango, formation but also to pattern the 18 lineage (see Section 10) Joel Rothman and Bill Wadsworth for sharing unpublished (Wang and Sternberg, 2000). data or communicating preprints. Our work is supported by grants from the EEC-TMR program, the Fondation pour la Recherche Me´dicale to GM, the Association pour la 11. Conclusions and perspectives Recherche contre le Cancer and the Ministe`re de la Recherche-ACI program to ML and by funds from the For a long time, the paucity of useful markers and the lack CNRS, INSERM and Hoˆpital Universitaire de Strasbourg. of well-defined phenotypes have hampered genetic analysis of morphogenesis in C. elegans. This gap is now being filled as the field is turning some of its attention to issues of cell References biology in development. In addition to the great collection of monoclonal antibodies against membrane markers devel- Albertson, D.G., Thomson, J.N., 1976. The pharynx of Caenorhabditis oped by Francis and Waterston (1991), which has been the elegans. Philos. Trans. R. Soc. Lond. B Biol. Sci. 275, 299–325. only source of markers for some time, a new set of GFP Arnold, H.H., Braun, T., 2000. Genetics of muscle determination and devel- fusions to several proteins localised in different subcellular opment. Curr. Top. Dev. Biol. 48, 129–164. compartments of epithelial cells is now available. In addi- Behrens, J., Mareel, M.M., Van Roy, F.M., Birchmeier, W., 1989. Dissect- tion, numerous mutations have been described that define ing tumor cell invasion: epithelial cells acquire invasive properties after the loss of uvomorulin-mediated cell-cell adhesion. J. Cell Biol. 108, phenotypes to score the different steps involved in a given 2435–2447. cellular process. Systematic genomic approaches, such as Bercher, M., Wahl, J., Vogel, B.E., Lu, C., Hedgecock, E.M., Hall, D.H., those made available by the RNA interference technology, Plenefisch, J.D., 2001. mua-3, a gene required for mechanical tissue global expression profiling and automated protein interac- integrity in Caenorhabditis elegans, encodes a novel transmembrane tion-mapping (Fraser et al., 2000; Gonczy et al., 2000; protein of epithelial attachment complexes. J. Cell. Biol. 154, 415–426. Berset, T., Hoier, E.F., Battu, G., Canevascini, S., Hajnal, A., 2001. Notch Reinke et al., 2000; Walhout et al., 2000), will further inhibition of RAS signaling through MAP kinase phosphatase LIP-1 increase the number of useful probes. With this in hand, C. during C. elegans vulval development. Science 291, 1055–1058. elegans should keep proving a powerful system to provide Bettinger, J.C., Euling, S., Rougvie, A.E., 1997. The terminal differentia- further insights into the mechanisms involved in connecting tion factor LIN-29 is required for proper vulval morphogenesis and egg cells and tissues together. As outlined above, one of the real laying in Caenorhabditis elegans. Development 124, 4333–4342. Bhat, M.A., Izaddoost, S., Lu, Y., Cho, K.O., Choi, K.W., Bellen, H.J., strengths of C. elegans lies in its genetic simplicity, which 1999. Discs Lost, a novel multi-PDZ domain protein, establishes and facilitates (sometimes eliminates) issues of genetic redun- maintains epithelial polarity. Cell 96, 833–845. dancy, as it is the case for laminin, integrin or intermediate Bilder, D., Birnbaum, D., Borg, J.P., Bryant, P., Huigbretse, J., Jansen, E., filament genes. In other cases, and rather unexpectedly as for Kennedy, M.B., Labouesse, M., Legouis, R., Mechler, B., Perrimon, N., apical junctions (CeAJs), C. elegans offers the possibility to Petit, M., Sinha, P., 2000a. Collective nomenclature for LAP proteins. Nat. Cell Biol. 2, E114. independently study functions which are intermingled in Bilder, D., Li, M., Perrimon, N., 2000b. Cooperative regulation of cell other species. One disadvantage of C. elegans to keep in polarity and growth by Drosophila tumor suppressors. Science 289, mind is the small size of certain cells (particularly epidermal 113–116. cells). In the near future, the field will certainly start integrat- Blelloch, R., Anna-Arriola, S.S., Gao, D., Li, Y., Hodgkin, J., Kimble, J., gon-1 Caenor- ing data from different areas, for instance examine how tran- 1999. The gene is required for gonadal morphogenesis in habditis elegans. Dev. Biol. 216, 382–393. scription control influences the expression of genes that Blelloch, R., Kimble, J., 1999. Control of organ shape by a secreted metal- orchestrate major morphogenetic changes. It will also loprotease in the nematode Caenorhabditis elegans. Nature 399, 586– begin exploring differential targeting in relation to lipid biol- 590. ogy, the complexity of the cytoskeleton, and how higher Borg, J.P., Marchetto, S., Le Bivic, A., Ollendorff, V., Jaulin-Bastard, F., order structures such as junctional complexes influence Saito, H., Fournier, E., Adelaide, J., Margolis, B., Birnbaum, D., 2000. ERBIN: a basolateral PDZ protein that interacts with the mammalian signalling. Caenorhabditis elegans occupies an ideal place ERBB2/HER2 receptor. Nat. Cell Biol. 2, 407–414. to do so, between unicellular organisms amenable to genetic Bossinger, O., Klebes, A., Segbert, C., Theres, C., Knust, E., 2001. Zonula 98 G. Michaux et al. / Gene 277 (2001) 83–100

adherens formation in Caenorhabditis elegans requires dlg-1, the Direct visualization of the elt-2 gut-specific GATA factor binding to homologue of the Drosophila gene discs large. Dev. Biol. 230, 29–42. a target promoter inside the living Caenorhabditis elegans embryo. Bredt, D.S., 1998. Sorting out genes that regulate epithelial and neuronal Proc. Natl. Acad. Sci. USA 96, 11883–11888. polarity. Cell 94, 691–694. Gatewood, B.K., Bucher, E.A., 1997. The mup-4 locus in Caenorhabditis Burke, R., Nellen, D., Bellotto, M., Hafen, E., Senti, K.A., Dickson, B.J., elegans is essential for hypodermal integrity, organismal morphogen- Basler, K., 1999. Dispatched, a novel sterol-sensing domain protein esis and embryonic body wall muscle position. Genetics 146, 165–183. dedicated to the release of cholesterol-modified hedgehog from signal- Gilleard, J.S., McGhee, J.D., 2001. Activation of hypodermal differentia- ing cells. Cell 99, 803–815. tion in the Caenorhabditis elegans embryo by GATA transcription Ch’ng, Q., Kenyon, C., 1999. egl-27 generates anteroposterior patterns of factors ELT-1 and ELT-3. Mol. Cell Biol. 21, 2533–2544. cell fusion in C. elegans by regulating Hox gene expression and Hox Gilleard, J.S., Shafi, Y., Barry, J.D., McGhee, J.D., 1999. ELT-3: A protein function. Development 126, 3303–3312. Caenorhabditis elegans GATA factor expressed in the embryonic Chang, C., Newman, A.P., Sternberg, P.W., 1999. Reciprocal EGF signal- epidermis during morphogenesis. Dev. Biol. 208, 265–280. ing back to the uterus from the induced C. elegans vulva coordinates Gissendanner, C.R., Sluder, A.E., 2000. nhr-25, the Caenorhabditis elegans morphogenesis of epithelia. Curr. Biol. 9, 237–246. orthologue of ftz-f1, is required for epidermal and somatic gonad devel- Chen, W., Lim, L., 1994. The Caenorhabditis elegans small GTP-binding opment. Dev. Biol. 221, 259–272. protein RhoA is enriched in the nerve ring and sensory neurons during Gonczy, P., Echeverri, G., Oegema, K., Coulson, A., Jones, S.J., Copley, larval development. J. Biol. Chem. 269, 32394–32404. R.R., Duperon, J., Oegema, J., Brehm, M., Cassin, E., Hannak, E., Chin-Sang, I.D., Chisholm, A.D., 2000. Form of the worm: genetics of Kirkham, M., Pichler, S., Flohrs, K., Goessen, A., Leidel, S., Alleaume, epidermal morphogenesis in C. elegans. Trends Genet. 16, 544–551. A.M., Martin, C., Ozlu, N., Bork, P., Hyman, A.A., 2000. Functional Clark, S.G., Chisholm, A.D., Horvitz, H.R., 1993. Control of cell fates in genomic analysis of cell division in C. elegans using RNAi of genes on the central body region of C. elegans by the homeobox gene lin-39. Cell chromosome III. Nature 408, 331–336. 74, 43–55. Greenwald, I., 1998. LIN-12/Notch signaling: lessons from worms and Costa, M., Raich, W., Agbunag, C., Leung, B., Hardin, J., Priess, J.R., 1998. flies. Genes Dev. 12, 1751–1762. A putative catenin-cadherin system mediates morphogenesis of the Hanna-Rose, W., Han, M., 1999. COG-2, a sox domain protein necessary Caenorhabditis elegans embryo. J. Cell Biol. 141, 297–308. for establishing a functional vulval-uterine connection in Caenorhab- Cruz, J.C., Sugii, S., Yu, C., Chang, T.Y., 2000. Role of Niemann-Pick type ditis elegans. Development 126, 169–179. C1 protein in intracellular trafficking of low density lipoprotein-derived Hawkins, M.G., McGhee, J.D., 1995. elt-2, a second GATA factor from the cholesterol. J. Biol. Chem. 275, 4013–4021. nematode Caenorhabditis elegans. J. Biol. Chem. 270, 14666–14671. Delattre, M., Felix, M.A., 1999. Connection of vulval and uterine epithelia Heid, P.J., Raich, W.B., Smith, R., Mohler, W.A., Simokat, K., Gendreau, in Caenorhabditis elegans. Biol. Cell 91, 573–583. S.B., Rothman, J.H., Hardin, J., 2001. The Zinc Finger Protein DIE-1 Is den Boer, B.G., Sookhareea, S., Dufourcq, P., Labouesse, M., 1998. A Required for Late Events during Epithelial Cell Rearrangement in C. tissue-specific knock-out strategy reveals that lin-26 is required for elegans. Dev. Biol. 236, 165–180. the formation of the somatic gonad epithelium in Caenorhabditis Herman, T., Hartwieg, E., Horvitz, H.R., 1999. sqv mutants of Caenorhab- elegans. Development 125, 3213–3224. ditis elegans are defective in vulval epithelial invagination. Proc. Natl. Dichtel, M.L., Louvet-Vallee, S., Viney, M.E., Felix, M.A., Sternberg, Acad. Sci. USA 96, 968–973. P.W., 2001. Control of vulval cell division number in the nematode Herman, T., Horvitz, H.R., 1999. Three proteins involved in Caenorhabdi- Oscheius/Dolichorhabditis sp. CEW1. Genetics 157, 183–197. tis elegans vulval invagination are similar to components of a glycosy- Doe, C.Q., Bowerman, B., 2001. Asymmetric cell division: fly neuroblast lation pathway. Proc. Natl. Acad. Sci. USA 96, 974–979. meets worm zygote. Curr. Opin. Cell Biol. 13, 68–75. Hermann, G.J., Leung, B., Priess, J.R., 2000. Left-right asymmetry in C. Drubin, D.G., Nelson, W.J., 1996. Origins of cell polarity. Cell 84, 335– elegans intestine organogenesis involves a LIN-12/Notch signaling 344. pathway. Development 127, 3429–3440. Eisenmann, D.M., Kim, S.K., 2000. Protruding vulva mutants identify Hoier, E.F., Mohler, W.A., Kim, S.K., Hajnal, A., 2000. The Caenorhab- novel loci and Wnt signaling factors that function during Caenorhab- ditis elegans APC-related gene apr-1 is required for epithelial cell ditis elegans vulva development. Genetics 156, 1097–1116. Eisenmann, D.M., Maloof, J.N., Simske, J.S., Kenyon, C., Kim, S.K., 1998. migration and Hox gene expression. Genes Dev. 14, 874–886. The beta-catenin homologue BAR-1 and LET-60 Ras coordinately Hong, L., Elbl, T., Ward, J., Franzini-Armstrong, C., Rybicka, K.K., Gate- regulate the Hox gene lin-39 during Caenorhabditis elegans vulval wood, B.K., Baillie, D.L., Bucher, E.A., 2001. MUP-4 is a novel trans- development. Development 125, 3667–3680. membrane protein with functions in epithelial cell adhesion in Eizinger, A., Sommer, R.J., 1997. The homeotic gene lin-39 and the evolu- Caenorhabditis elegans. J. Cell Biol. 154, 403–414. tion of nematode epidermal cell fates. Science 278, 452–455. Horvitz, H.R., Sulston, J.E., 1980. Isolation and genetic characterisation of Fay, D.S., Han, M., 2000. The synthetic multivulval genes of C. elegans: cell-lineage mutants of the nematode Caenorhabditis elegans. Genetics functional redundancy, Ras-antagonism, and cell fate determination. 96, 435–454. Genesis 26, 279–284. Hoskins, R., Hajnal, A.F., Harp, S.A., Kim, S.K., 1996. The C. elegans Felix, M.A., Sternberg, P.W., 1997. Two nested gonadal inductions of the vulval induction gene lin-2 encodes a member of the MAGUK family of vulva in nematodes. Development 124, 253–259. cell junction proteins. Development 122, 97–111. Francis, R., Waterston, R.H., 1991. Muscle cell attachment in Caenorhab- Hresko, M.C., Schriefer, L.A., Shrimankar, P., Waterston, R.H., 1999. ditis elegans. J. Cell Biol. 114, 465–479. Myotactin, a novel hypodermal protein involved in muscle-cell adhe- Fraser, A.G., Kamath, R.S., Zipperlen, P., Martinez-Campos, M., Sohr- sion in Caenorhabditis elegans. J. Cell Biol. 146, 659–672. mann, M., Ahringer, J., 2000. Functional genomic analysis of C. Hresko, M.C., Williams, B.D., Waterston, R.H., 1994. Assembly of body elegans chromosome I by systematic RNA interference. Nature 408, wall muscle and muscle cell attachment structures in Caenorhabditis 325–330. elegans. J. Cell Biol. 124, 491–506. Fuchs, E., Cleveland, D.W., 1998. A structural scaffolding of intermediate Hubbard, E.J., Greenstein, D., 2000. The Caenorhabditis elegans gonad: a filaments in health and disease. Science 279, 514–519. test tube for cell and developmental biology. Dev. Dyn. 218, 2–22. Fukushige, T., Hawkins, M.G., McGhee, J.D., 1998. The GATA-factor elt- Hutter, H., Vogel, B.E., Plenefisch, J.D., Norris, C.R., Proenca, R.B., 2 is essential for formation of the Caenorhabditis elegans intestine. Spieth, J., Guo, C., Mastwal, S., Zhu, X., Scheel, J., Hedgecock, Dev. Biol. 198, 286–302. E.M., 2000. Conservation and novelty in the evolution of cell adhesion Fukushige, T., Hendzel, M.J., Bazett-Jones, D.P., McGhee, J.D., 1999. and extracellular matrix genes. Science 287, 989–994. G. Michaux et al. / Gene 277 (2001) 83–100 99

Hynes, R.O., 1999. Cell adhesion: old and new questions. Trends Cell Biol. mere by the combined action of SKN-1 and a GSK-3b homologue is 9, M33–M37. mediated by MED-1 and -2 in C. elegans. Mol. Cell 7, 475–485. Ikonen, E., Simons, K., 1998. Protein and lipid sorting from the trans-Golgi McMahon, L., Legouis, R., Vonesch, J.-L., Labouesse, M., 2001. Assembly network to the plasma membrane in polarised cells. Sem. Cell Dev. of C. elegans apical junctions involves positioning and compaction by Biol. 9, 503–509. LET-413 and protein aggregation by the MAGUK protein DLG-1. J. Itoh, M., Morita, K., Tsukita, S., 1999. Characterization of ZO-2 as a Cell Sci. 114, 2265–2277. MAGUK family member associated with tight as well as adherens Mellman, I., Warren, G., 2000. The road taken: past and future foundations junctions with a binding affinity to occludin and alpha catenin. J. of membrane traffic. Cell 100, 99–112. Biol. Chem. 274, 5981–5986. Meneghini, M.D., Ishitani, T., Carter, J.C., Hisamoto, N., Ninomiya-Tsuji, Kaech, S.M., Whitfield, C.W., Kim, S.K., 1998. The LIN-2/LIN-7/LIN-10 J., Thorpe, C.J., Hamill, D.R., Matsumoto, K., Bowerman, B., 1999. complex mediates basolateral membrane localisation of the C. elegans MAP kinase and Wnt pathways converge to downregulate an HMG- EGF receptor LET-23 in vulval epithelial cells. Cell 94, 761–771. domain repressor in Caenorhabditis elegans. Nature 399, 793–797. Kaletta, T., Schnabel, H., Schnabel, R., 1997. Binary specification of the Metzger, R.J., Krasnow, M.A., 1999. Genetic control of branching morpho- embryonic lineage in Caenorhabditis elegans. Nature 390, 294–298. genesis. Science 284, 1635–1639. Karabinos, A., Schmidt, H., Harborth, J., Schnabel, R., Weber, K., 2001. Michaux, G., Gansmuller, A., Hindelang, C., Labouesse, M., 2000. CHE- Essential roles for four cytoplasmic intermediate filament proteins in 14, a protein with a sterol-sensing domain, is required for apical sorting Caenorhabditis elegans development. Proc. Natl. Acad. Sci. USA 98, in C. elegans ectodermal epithelial cells. Curr. Biol. 10, 1098–1107. 7863–7868. Miyabayashi, T., Palfreyman, M.T., Sluder, A.E., Slack, F., Sengupta, P., Kim, S.K., Johansson, A., Driessens, M., Aspenstrom, P., 2000. Cell polar- 1999. Expression and function of members of a divergent nuclear recep- ity: new PARtners for Cdc42 and Rac. Nat. Cell Biol. 2, E143–E145. tor family in Caenorhabditis elegans. Dev. Biol. 215, 314–331. Kimble, J., 1981. Alterations in cell lineage following laser ablation of cells Moghal, N., Sternberg, P.W., 1999. Multiple positive and negative regula- in the somatic gonad of Caenorhabditis elegans. Dev. Biol. 87, 286– tors of signaling by the EGF- receptor. Curr. Opin. Cell Biol. 11, 190– 300. 196. Klebes, A., Knust, E., 2000. A conserved motif in Crumbs is required for E- Mohler, W.A., Simske, J.A., Williams-Masson, E.M., Hardin, J.D., White, cadherin localisation and zonula adherens formation in Drosophila. J.G., 1998. Dynamics and ultrastructure of developmental cells fusions Curr. Biol. 10, 76–85. in the Caenorhabditis elegans hypodermis. Curr. Biol. 8, 1087–1090. Koeppen, M., Simske, J.S., Sims, P.A., Firestein, B.L., Hall, D.H., Radice, Muller, H.A., 2000. Genetic control of epithelial cell polarity: lessons from A.D., Rongo, C., Hardin, J.D., 2001. Cooperative regulation of AJM-1 Drosophila. Dev. Dyn. 218, 52–67. by Discs large and LET-413 controls junctional integrity of Caenor- Neufeld, E.B., Wastney, M., Patel, S., Suresh, S., Cooney, A.M., Dwyer, habditis elegans epithelia. Nat. Cell Biol. (in press). N.K., Roff, C.F., Ohno, K., Morris, J.A., Carstea, E.D., Incardona, J.P., Koh, K., Rothman, J.H., 2001. ELT-5 and ELT-6. Development 128, 2867– Strauss 3rd, J.F., Vanier, M.T., Patterson, M.C., Brady, R.O., Pentchev, 2880. P.G., Blanchette-Mackie, E.J., 1999. The Niemann-Pick C1 protein Korswagen, H.C., Herman, M.A., Clevers, H.C., 2000. Distinct beta-cate- resides in a vesicular compartment linked to retrograde transport of nins mediate adhesion and signalling functions in C. elegans. Nature multiple lysosomal cargo. J. Biol. Chem. 274, 9627–9635. 406, 527–532. Newman, A.P., Acton, G.Z., Hartwieg, E., Horvitz, H.R., Sternberg, P.W., Kostrouchova, M., Krause, M., Kostrouch, Z., Rall, J.E., 1998. CHR3: a 1999. The lin-11 LIM domain transcription factor is necessary for Caenorhabditis elegans orphan nuclear hormone receptor required for morphogenesis of C. elegans uterine cells. Development 126, 5319– proper epidermal development and molting. Development 125, 1617– 5326. 1626. Newman, A.P., Inoue, T., Wang, M., Sternberg, P.W., 2000. The Caenor- Kramer, J.M., 1997. Extracellular matrix. In: Riddle, D.L., Blumenthal, T., habditis elegans heterochronic gene lin-29 coordinates the vulval-uter- Meyer, B.J., Priess, J.R. (Eds.), C elegans II. Cold Spring Harbor ine-epidermal connections. Curr. Biol. 10, 1479–1488. Laboratory Press, Cold Spring Harbor, NY, pp. 471–500. Newman, A.P., Sternberg, P.W., 1996. Coordinated morphogenesis of Kuchinke, U., Grawe, F., Knust, E., 1998. Control of spindle orientation in epithelia during development of the Caenorhabditis elegans uterine- Drosophila by the Par-3-related PDZ-domain protein Bazooka. Curr. vulval connection. Proc. Natl. Acad. Sci. USA 93, 9329–9333. Biol. 8, 1357–1365. Newman, A.P., White, J.G., Sternberg, P.W., 1995. The Caenorhabditis Labouesse, M., 1997. Deficiency screen based on the monoclonal antibody elegans lin-12 gene mediates induction of ventral uterine specialization MH27 to identify genetic loci required for morphogenesis of the by the anchor cell. Development 121, 263–271. Caenorhabditis elegans embryo. Dev. Dyn. 210, 19–32. Newman-Smith, E.D., Rothman, J.H., 1998. The maternal-to-zygotic tran- Labouesse, M., Hartwieg, E., Horvitz, H.R., 1996. The Caenorhabditis sition in embryonic patterning of Caenorhabditis elegans. Curr. Opin. elegans LIN-26 protein is required to specify and/or maintain all non- Genet. Dev. 8, 472–480. neuronal ectodermal cell fates. Development 122, 2579–2588. Nishiwaki, K., Hisamoto, N., Matsumoto, K., 2000. A metalloprotease Labouesse, M., Mango, S.E., 1999. Patterning the C. elegans embryo: disintegrin that controls cell migration in Caenorhabditis elegans. moving beyond the cell lineage. Trends Genet. 15, 307–313. Science 288, 2205–2208. Labouesse, M., Sookhareea, S., Horvitz, H.R., 1994. The Caenorhabditis Noselli, S., Agnes, F., 1999. Roles of the JNK signaling pathway in Droso- elegans gene lin-26 is required to specify the fates of hypodermal cells phila morphogenesis. Curr. Opin. Genet. Dev. 9, 466–472. and encodes a presumptive zinc-finger transcription factor. Develop- Page, B.D., Zhang, W., Steward, K., Blumenthal, T., Priess, J.R., 1997. ment 120, 2359–2368. ELT-1, a GATA-like transcription factor, is required for epidermal cell Lange, Y., Steck, T.L., 1998. Four cholesterol-sensing proteins. Curr. Opin. fates in Caenorhabditis elegans embryos. Genes Dev. 11, 1651–1661. Struct. Biol. 8, 435–439. Petronczki, M., Knoblich, J.A., 2001. DmPAR-6 directs epithelial polarity Legouis, R., Gansmuller, A., Sookhareea, S., Bosher, J.M., Baillie, D.L., and asymmetric cell division of neuroblasts in Drosophila. Nat. Cell Labouesse, M., 2000. LET-413 is a basolateral protein required for the Biol. 3, 43–49. assembly of adherens junctions in Caenorhabditis elegans. Nat. Cell Plenefisch, J.D., Zhu, X., Hedgecock, E.M., 2000. Fragile skeletal muscle Biol. 2, 415–422. attachments in dystrophic mutants of Caenorhabditis elegans: isolation Leung, B., Hermann, G.J., Priess, J.R., 1999. Organogenesis of the Caenor- and characterisation of the mua genes. Development 127, 1197–1207. habditis elegans intestine. Dev. Biol. 216, 114–134. Portereiko, M.F., Mango, S.E., 2001. Early morphogenesis of the C. Maduro, M.F., Meneghini, M.D., Bowerman, B., Broitman-Maduro, G., elegans pharynx. Dev. Biol. 233, 482–494. Rothman, J.H., 2001. Restriction of mesendoderm to a single blasto- Priess, J.R., Hirsh, D.I., 1986. Caenorhabditis elegans morphogenesis: the 100 G. Michaux et al. / Gene 277 (2001) 83–100

role of the cytoskeleton in elongation of the embryo. Dev. Biol. 117, molecules, nectin and cadherin, interact through their cytoplasmic 156–173. domain-associated proteins. J. Cell Biol. 150, 1161–1176. Quintin, S., Michaux, G., McMahon, L., Gansmuller, A., Labouesse, M., Thiery, J.P., Boyer, B., Tucker, G., Gavrilovic, J., Valles, A.M., 1988. 2001. The C. elegans gene lin-26 can trigger epithelial differentiation Adhesion mechanisms in embryogenesis and in cancer invasion and without conferring tissue specificity. Dev. Biol. 235, 410–421. metastasis. Ciba Foundation Symp. 141, 48–74. Raich, W.B., Agbunag, C., Hardin, J., 1999. Rapid epithelial-sheet sealing Tsukita, S., Furuse, M., Itoh, M., 1999. Structural and signalling molecules in the Caenorhabditis elegans embryo requires cadherin-dependent filo- come together at tight junctions. Curr. Opin. Cell Biol. 11, 628–633. podial priming. Curr. Biol. 9, 1139–1146. Vogel, B.E., Hedgecock, E.M., 2001. Hemicentin, a conserved extracellular Reinke, V., Smith, H.E., Nance, J., Wang, J., Van Doren, C., Begley, R., member of the immunoglobulin superfamily, organises epithelial and Jones, S.J., Davis, E.B., Scherer, S., Ward, S., Kim, S.K., 2000. A other cell attachments into oriented line-shaped junctions. Development global profile of germline gene expression in C. elegans. Mol. Cell 6, 128, 883–894. 605–616. Walhout, A.J., Sordella, R., Lu, X., Hartley, J.L., Temple, G.F., Brasch, Reiter, J.F., Alexander, J., Rodaway, A., Yelon, D., Patient, R., Holder, N., M.A., Thierry-Mieg, N., Vidal, M., 2000. Protein interaction mapping Stainier, D.Y., 1999. Gata5 is required for the development of the heart in C. elegans using proteins involved in vulval development. Science and endoderm in zebrafish. Genes Dev. 13, 2983–2995. 287, 116–122. Reuter, R., 1994. The gene serpent has hoemotic properties and specifies Wang, A.Z., Ojakian, G.K., Nelson, W.J., 1990a. Steps in the morphogen- endoderm versus ectoderm within the Drosophila gut. Development esis of a polarised epithelium. I. Uncoupling the roles of cell-cell and 120, 1123–1135. cell-substratum contact in establishing plasma membrane polarity in Rocheleau, C.E., Downs, W.D., Lin, R., Wittmann, C., Bei, Y., Cha, Y.-H., multicellular epithelial (MDCK) cysts. J. Cell Sci. 95, 137–151. Ali, M., Priess, J.R., Mello, C.C., 1997. Wnt signaling and an APC- Wang, A.Z., Ojakian, G.K., Nelson, W.J., 1990b. Steps in the morphogen- related gene specify endoderm in early C. elegans embryos. Cell 90, esis of a polarised epithelium. II. Disassembly and assembly of plasma 707–716. membrane domains during reversal of epithelial cell polarity in multi- Rocheleau, C.E., Yasuda, J., Shin, T.H., Lin, R., Sawa, H., Okano, H., cellular epithelial (MDCK) cysts. J. Cell Sci. 95, 153–165. Priess, J.R., Davis, R.J., Mello, C.C., 1999. WRM-1 activates the Wang, M., Sternberg, P.W., 2000. Patterning of the C. elegans 1 degrees vulval lineage by RAS and wnt pathways. Development 127, 5047– LIT-1 protein kinase to transduce anterior/posterior polarity signals in 5058. C. elegans. Cell 97, 717–726. White, J., 1988. The anatomy. In: Wood, W.W., the community of C. Rose, L.S., Kemphues, K.J., 1998. Early patterning of the C. elegans elegans researchers (Eds.), The Nematode Caenorhabditis elegans. embryo. Ann. Rev. Genet. 32, 521–545. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. Salser, S.J., Loer, C.M., Kenyon, C., 1993. Multiple HOM-C gene interac- 81–122. tions specify cell fates in the nematode central nervous system. Genes Whitfield, C.W., Benard, C., Barnes, T., Hekimi, S., Kim, S.K., 1999. Dev. 7, 1714–1724. Basolateral localisation of the Caenorhabditis elegans epidermal Scheel, J., Srinivasan, J., Honnert, U., Henske, A., Kurzchalia, T.V., 1999. growth factor receptor in epithelial cells by the PDZ protein LIN-10. Involvement of caveolin-1 in meiotic cell-cycle progression in Caenor- Mol. Biol. Cell. 10, 2087–2100. habditis elegans. Nat. Cell Biol. 1, 127–129. Williams, B.D., Waterston, R.H., 1994. Genes critical for muscle develop- Schober, M., Schaefer, M., Knoblich, J.A., 1999. Bazooka recruits Inscute- ment and function in Caenorhabditis elegans identified through lethal able to orient asymmetric cell divisions in Drosophila neuroblasts. mutations. J. Cell Biol. 124, 475–490. Nature 402, 548–551. Wissmann, A., Ingles, J., Mains, P.E., 1999. The Caenorhabditis elegans Sharma-Kishore, R., White, J.G., Southgate, E., Podbilewicz, B., 1999. mel-11 myosin phosphatase regulatory subunit affects tissue contrac- Formation of the vulva in Caenorhabditis elegans: a paradigm for tion in the somatic gonad and the embryonic epidermis and genetically organogenesis. Development 126, 691–699. interacts with the Rac signaling pathway. Dev. Biol. 209, 111–127. Shemer, G., Kishore, R., Podbilewicz, B., 2000. Ring formation drives Wissmann, A., Ingles, J., McGhee, J.D., Mains, P.E., 1997. Caenorhabditis invagination of the vulva in Caenorhabditis elegans: Ras, cell fusion, elegans LET-502 is related to Rho-binding kinases and human and cell migration determine structural fates. Dev. Biol. 221, 233–248. myotonic dystrophy kinase and interacts genetically with a homologue Shemer, G., Podbilewicz, B., 2000. Fusomorphogenesis: cell fusion in of the regulatory subunit of smooth muscle myosin phosphatase to organ formation. Dev. Dyn. 218, 30–51. affect cell shape. Genes Dev. 11, 409–422. Shoichet, S.A., Malik, T.H., Rothman, J.H., Shivdasani, R.A., 2000. Action Wodarz, A., Ramrath, A., Grimm, A., Knust, E., 2000. Drosophila atypical of the Caenorhabditis elegans GATA factor END-1 in Xenopus protein kinase C associates with Bazooka and controls polarity of suggests that similar mechanisms initiate endoderm development in epithelia and neuroblasts. J. Cell Biol. 150, 1361–1374. ecdysozoa and vertebrates. Proc. Natl. Acad. Sci. USA 97, 4076–4081. Yap, A.S., Brieher, W.M., Gumbiner, B.M., 1997. Molecular and func- Simske, J.S., Hardin, J., 2001. Getting into shape: epidermal morphogenesis tional analysis of cadherin-based adherens junctions. Ann. Rev. Cell in Caenorhabditis elegans embryos. BioEssays 23, 12–23. Dev. Biol. 13, 119–146. Simske, J.S., Kaech, S.M., Harp, S.A., Kim, S.K., 1996. LET-23 receptor Yeaman, C., Grindstaff, K.K., Nelson, W.J., 1999. New perspectives on localisation by the cell junction protein LIN-7 during C. elegans vulval mechanisms involved in generating epithelial cell polarity. Physiol. induction. Cell 85, 195–204. Rev. 79, 73–98. Steinert, P.M., Cantieri, J.S., Teller, D.C., Lonsdale-Eccles, J.D., Dale, Yochem, J., Tuck, S., Greenwald, I., Han, M., 1999. A gp330/megalin- B.A., 1981. Characterization of a class of cationic proteins that speci- related protein is required in the major epidermis of Caenorhabditis fically interact with intermediate filaments. Proc. Natl. Acad. Sci. USA elegans for completion of molting. Development 126, 597–606. 78, 4097–4101. Zhu, J., Fukushige, T., McGhee, J.D., Rothman, J.H., 1998. Reprogram- Sternberg, P.W., Han, M., 1998. Genetics of RAS signaling in C. elegans. ming of early embryonic blastomeres into endodermal progenitors by a Trends Genet. 14, 466–472. Caenorhabditis elegans GATA factor. Genes Dev. 12, 3809–3814. Sternberg, P.W., Horvitz, H.R., 1986. Pattern formation during vulval Zhu, J., Hill, R.J., Heid, P.J., Fukuyama, M., Sugimoto, A., Priess, J.R., development in C. elegans. Cell 44, 761–772. Rothman, J.H., 1997. end-1 encodes an apparent GATA factor that Tachibana, K., Nakanishi, H., Mandai, K., Ozaki, K., Ikeda, W., Yama- specifies the endoderm precursor in Caenorhabditis elegans embryos. moto, Y., Nagafuchi, A., Tsukita, S., Takai, Y., 2000. Two cell adhesion Genes Dev. 11, 2883–2896.