From A to Z: apical structures and zona pellucida-domain proteins

Review

From A to Z: apical structures and zona pellucida-domain proteins Serge Plaza1,3, He´le`ne Chanut-Delalande1,3, Isabelle Fernandes1,3*, Paul M. Wassarman2 and Franc¸ois Payre1,3 1

Universite´ de Toulouse, UPS; Centre de Biologie du De´veloppement; F-31062, Toulouse, France Department of Developmental and Regenerative Biology, Mount Sinai School of Medicine, 1468 Madison Avenue, New York, NY 10029, USA 3 CNRS, UMR5547, Centre de Biologie du De´veloppement, F-31062, Toulouse, France 2

The terminal differentiation of epithelial cells involves changes in the apical compartment, including remodeling of the cytoskeleton and junctions to modify its threedimensional organization. It also often triggers the building of specialized extracellular matrices, the function of which remains poorly understood. Hundreds of extracellular matrix proteins expressed in a variety of epithelia possess a conserved region called the zona pellucida-domain (ZP domain). There is evidence to suggest that ZP-domains mediate the polymerization of proteins into fibrils or matrices and that mutation of ZP-domains can result in severe pathologies, such as infertility, deafness, and cancer. Recent work in worms and flies demonstrates that ZP-domain proteins play a crucial role in organizing and shaping highly specialized apical structures in epithelial cells. Introduction The function of many differentiated cells relies on a polarized organization. This includes cell processes and extensions that locally transform cell shape, often at the apical surface. Remodeling of the apical compartment is crucial for morphogenesis of epithelial cells and tissues during development. Recent work sheds new light on the apical extracellular matrix (ECM), demonstrating the importance of zona pellucida-domain (ZP-domain) proteins in the organization of highly specialized apical structures. Founding members of the zona pellucida (ZP) family of proteins were first identified as components of the extracellular coat, or ZP, of mammalian ova [1,2]. There are three or more ZP proteins present in the extracellular coat of mammalian ova and these form a dense network of fibers as a result of heterotypic ‘and possibly homotypic’ interactions [3,4]. The association between ZP proteins relies on the presence of a common motif, called the ZP-domain [1,5,6]. It is now recognized that a ZP-domain is present in many proteins that are expressed in differentiated epithelia and neural tissues. Most ZP-domain proteins are subject to extensive post-translational modifications, including proteolytic processing and glycosylation with asparagine- (N-) and/or serine/threonine- (O-) linked oligosaccharides. Consequently, their molecular complexity has Corresponding author: Plaza, S. ([email protected]) Present address: Department of Biology, McGill University, 1205 Dr. Penfield Avenue, Montreal, QC, Canada H3A 1B1. *

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made it difficult to determine the three-dimensional structure of ZP-domain proteins. Furthermore, cell lines generally do not produce highly differentiated phenotypes, such as the formation of proper ECM, therefore hampering the functional characterization of ZP-domain proteins in ex vivo systems. In the past 10 years or so, genetic analyses in model systems, such as worms and flies, have revealed roles for ZP-domain proteins in the differentiation of various developing tissues. In addition, mapping human genes involved in diseases has shown that several ZP-domain proteins impact on human physiology. From genetic studies it has emerged that ZP-domain proteins play a role in the control of cell shape. Recent work suggests that ZP-domain proteins are involved in remodeling the apical organization of various cells during morphological differentiation. This review summarizes recent advances made in our understanding of ZP-domain protein function, and focuses on their role during differentiation of epithelial tissues. Molecular organization of ZP-domain proteins Although proteins possessing a ZP-domain are involved in a wide range of biological functions, they share certain common features. For example, in addition to having a ZPdomain, these proteins usually possess a signal peptide, a conserved stretch of basic amino acid residues that precedes a single transmembrane domain, and a short cytoplasmic tail (Figure 1A). The ZP-domain is composed of 260 amino acids and has eight, ten (or 12) conserved Cys residues that define a ZP-domain signature. These Cys residues are present in four to six disulfide bonds that are generally well conserved [7–9], although there is evidence of variations in Cys linkage between ZP proteins of different species [10]. ZP-domain proteins often form long interconnected fibrils or filaments that exhibit a repeated structure [11,12]. For example, this is the case for the mammalian ZP, where homodimers of ZP1 crosslink filaments formed by ZP2/ZP3 polymers [5], as well as for the vitelline envelope proteins VEbeta and VEgamma that play a similar role in the trout [3]. Uromodulin, another ZP-domain protein, forms fibrils with a repeated zig-zag course, which merge into larger bundles [5,13]. The isolated uromodulin ZPdomain is sufficient to promote formation of similar fibrils, suggesting that the ZP-domain acts as a polymerization

0962-8924/$ – see front matter ß 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.tcb.2010.06.002 Trends in Cell Biology 20 (2010) 524–532

Review module [13,14]. The ZP-domain is actually a module consisting of two domains, called ZP-N (N-terminal) and ZP-C (C-terminal), separated by a short linker region [(Figure_1)TD$IG](Figure 1A). A small number of proteins contain only the

Figure 1. Structural features of ZP-domain proteins. (a) Schematic representation of the overall organization of an archetypical ZP-domain protein. Shown are the Nterminal signal peptide (SP, red), the ZP-domain (pink) and a transmembrane region (TM, blue). It should be noted that some ZP-domain proteins possess a GPI anchor; see Jovine et al. for an extensive review [5]. The ZP-domain possesses highly conserved Cys residues and consists of two regions, ZP-N and ZP-C, each with specific disulfide bonding. A tyrosine residue (Y) well conserved in the ZP-N region participates in ZP-domain folding; its substitution by a Cys residue impairs both polymerization [16] and in vivo function of ZP-domain proteins, in mammals [17] and flies [18]. Additional Cys residues present in the ZP-C portion of some ZPdomains are indicated in green. Also shown are the positions of the basic putative cleavage site (PCS), and additional domains (green) present in various ZP-domain proteins. (b) Representation of the three-dimensional structure of the ZP-N domain of mouse ZP3. Rainbow-colored regions from blue to red color code the N-terminal to C-terminal, respectively (taken from [16]). The tyrosine residue underlined in part (a) corresponds to Y 111.

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ZP-N domain [15] and the latter appears sufficient to form polymers in vitro [14,16]. Among ZP-domain proteins, there are at least four alternative disulfide linkages within the ZP-C region [7]; these involve either four, six, or eight Cys residues, a feature that could be related to differences in the mode of protein interactions [5]. Recently, the three-dimensional structure of the ZP-N domain of mouse ZP3 has been determined at high resolution (see Figure 1B) and the structure represents a new subtype of the immunoglobulin (IgG) family [16]. This work provides support for the important role of disulfide bonds in ZP-domain proteins and highlights the contribution of other residues in ZP-domain folding. Two of these residues, Tyr111 and Tyr141, are well conserved among species and ensure aromatic interactions that contribute to proper folding of ZP-N [16]. In this context, a point mutation converting Tyr111 to Cys in the ZP-domain of human atectorin, another ZP-domain protein, leads to a dominant negative phenotype (Tyr1870Cys mutation of a-tectorin) [17]. Introducing this mutation into mouse protein ZP3 impairs its secretion and polymerization properties [16]. Recent in vivo analyses in flies indicate that the functional organization of ZP-N is conserved throughout species. The same Tyr to Cys substitution compromises the function of the ZP-domain protein Miniature (M) during Drosophila development. Furthermore, this mutated form, Mtecta, exerts a dominant effect when expressed in wild-type animals, suggesting that alteration of ZP-N structure affects polymerization of ZP-domain proteins [18]. Many ZP-domain proteins possess additional motifs or domains between the N-terminal signal peptide and the ZP-domain (Figure 1A). These include plasminogen activator N-terminal (PAN), Epidermal growth factor (EGF and EGF-like), von Willebrand factor (vWA), scavenger receptor cysteine rich (SRCR), and entactin domains [5]. Mutations in the vWA domain of a-tectorin compromise its activity, emphasizing the importance of this domain for the in vivo function of the protein [17]. Furthermore, mutations affecting human uromodulin are often located in the EGF-like domains of this protein [19,20]. Genetic assays in Drosophila have permitted examination of the respective contribution of the ZP-domain and other regions in the activity of a subfamily of ZP-domain proteins. Both their ZP-domain and PAN motifs are responsible for the functional specificity of these proteins. Moreover swapping the ZP-domain of one protein with that of one of its paralogs, or transferring PAN domains, is not sufficient to efficiently switch their in vivo activities [18]. In addition, the region between the ZP- and transmembrane domains also participates in functional specificity. In a similar vein, an extracellular region located downstream of the ZPdomain is responsible for the species-restricted specificity of ZP3 function during fertilization in mice [21]. Therefore, although the ZP-domain represents a crucial feature of ZPdomain proteins, their functional diversification during evolution has relied on the inclusion of additional domains and sequences, as well as on modifications of the juxtamembrane extracellular region. ZP-domain proteins exhibit different degrees of posttranslational modifications and the effect of these has remained difficult to assess. For example, ZP-domain 525

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proteins are often synthesized as precursors that undergo proteolytic cleavage with release of the bulk of the protein into the extracellular space. As indicated in Figure 1A, there is a conserved stretch of basic residues close to the transmembrane region of most ZP-domain proteins that serves as a potential cleavage-site for (a) furin-like protease(s) [22,23]. However, substituting these basic residues does not entirely prevent proteolytic processing of ZP proteins by other proteases and release of the extracellular moiety [24–26]. Cleavage of ZP-domain proteins also occurs in epithelial cells, as exemplified by a cleaved form of uromodulin that is excreted by renal cells [27]. Although uromodulin is a glycosyl-phosphatidylinositol (GPI) anchored protein [28], Piopio (Pio) in flies and DYF-7 in worms each possess a transmembrane region and are found as processed proteins lacking their C-terminal region [29,30]. In the latter case, whereas substitution of putative furin cleavage sites inhibits release of the extracellular region, this modified protein is still able to compensate for the lack of endogenous product when reintroduced into whole animals [30]. Therefore, although several ZP-domain proteins apparently undergo maturation via proteolytic cleavage, the functional importance of this cleavage is not always clear. In summary, ZP-domain proteins are extracellular modular proteins that display a conserved three-dimensional structure. These proteins can be anchored to the mem-

brane or released into the extracellular space. Their biochemical properties suggest that they play a role in ECM organization. ZP-domain proteins in development As in mammalian fertilization, ZP-domain proteins also participate during fertilization in many non-mammalian species [31]; this includes fishes where ZP-domain proteins are often synthesized by somatic tissues. It is now clear that ZP-domain proteins fulfill a wide range of additional biological functions (Table 1). In invertebrates possessing a cuticle (e.g., worms and flies) several ZP-domain proteins are present as components of this exoskeleton. CUT-1, CUT-3, CUT-4, CUT-5, and CUT-6 are ZP-domain proteins that are coexpressed in a specific subset of hypodermal cells, triggering formation of the alae (Figure 2A and Tables 1 and 2) [32–34], which are longitudinal extroflessions of the cuticle that runs bilaterally along the body of the worm. Genomic analysis has revealed that two ZP-domain proteins, NOAH-1 and -2, are required for molting [35]. Male worms lacking the RAM-5 ZP-domain protein display misshaped sensory rays, a phenotype resulting from abnormal morphogenesis of hypodermal cells (Table 1) [36]. In Drosophila, 18 genes encode ZP-domain proteins that are often expressed in cuticle secreting cells [37]. Several of these Drosophila ZP-domain proteins are involved in the

Table 1. ZP-domain proteins involved in cell morphogenesis Sp. mammals

Name (synonym) Tectorin a (TECTA) Tectorin b Hensin (CRP, ductin, DMBT-1, GP-340) Uromodulin (UMOD, THP) Endoglin (CD105) b-Glycan (TGFBR3)

drosophila

nematode

Protein motifs SP, partial entactin G1 vWA, ZP-domain, GPI anchor SP, ZP-domain, GPI anchor SP, SRCR, CUB, ZP-domain

Distribution Tectorial membrane of cochlear cells

Diseases/developmental defects Deafness

ECM of various epithelial cells

Cancers

[52,62]

SP, EGF/EGF-like, ZP-domain, GPI anchor SP, ZP-domain, TMD

Kidney cells, secreted in urine Apical surface of vascular cells Apical surface of various epithelia

Renal diseases, urinary tract infections Cancers

[19,20,88,89]

Tip of mechanosensory sense organs aECM of tracheal and epidermal cells aECM of epidermal cells

Touch insensitive, hearing loss

[42]

Tracheal defects, wing blisters

[29,39]

Small wings, abnormal shape of denticle cells

[18,38,40]

aECM of embryonic epidermal cells

Abnormal shape of denticle cells

[18,37,38]

Abnormal shape of alae (L1, dauer stages) Abnormal shape of alae, dumpy worms Larval death, excretory defects Molting defects Lumpy, amorphous rays Dendrite extension defects

[33,34]

SP, ZP-domain, TMD

NompA

SP, PAN, ZP-domain, TMD

Piopio Dumpy Miniature

SP, ZP-domain, TMD EGF, ZP-domain, TMD SP, ZP-domain, TMD

Dusky Zye

ZP-domain, TMD SP, ZP-domain

Dusky-like Trynity Neyo, Nyobe, Morpheyus

SP, ZP-domain, TMD PAN, ZP-domain, TMD SP, PAN, ZP-domain, TMD

CUT-1, CUT-3, CUT-4, CUT-5 CUT-6

SP, ZP-domain, TMD

aECM of hypodermal cells

SP, vWA, ZP-domain, TMD

aECM of hypodermal cells

LET-653 NOAH-1 & 2 RAM-5 DYF-7

SP, PAN, ZP-domain, TMD SP,PAN, ZP-domain, TMD ZP-domain, TMD SP, ZP-domain, TMD

Apical surface of epithelia unknown Sensory support cells Tip of amphid sense organs

Ref. [17,44,45,47]

[61]

[33]

[68] [35] [36] [30]

SP, signal peptide; PAN, PlAsminogen N terminus domain; vWA, von Willebrand Factor; CUB, Complement subcomponents Clr/s, Uegf protein, and Bone morphogenetic protein; SRCR, scavenger receptor cysteine rich; ZP-domain, Zona Pellucida domain; TMD, transmembrane domain; GPI, glycosyl phosphatidylinositol.

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Figure 2. ZP-domain proteins control epithelial cell shape. (a): Inactivation of ZP-domain genes results in morphological defects in C. elegans. The alae, which are bilateral cuticle structures along the developing worm body, are not properly formed, or are missing in the larva upon inactivation of cut-1 and cut-6, two genes that encode ZPdomain proteins. Inactivation of the ZP-domain cut-4 gene alters alae in adults (micrographs are taken from [33]). (b) Scanning electron micrographs of the apical surface of rabbit kidney intercalated cells seeded at low (left) and high densities (right). Cells seeded at high density induce hensin expression and subsequent polymerization, which switch their three-dimensional organization from a flattened to columnar morphology harboring numerous microvilli at their apical side (villin in red, nuclei in green) (taken from [87]). (c) Loss-of-function of ZP-domain genes leads to abnormal apical cell extensions (denticles) in epidermal cells of the Drosophila embryo. When compared with denticles formed in wild-type embryos (wt), the absence of the Miniature or Dusky-like ZP-domain proteins in the corresponding mutants (m or dyl) leads to specific defects in the shape of the denticles, owing to localized detachment of the aECM (SEM pictures taken from [18]). (d) Apical cell junctions, visualized with a transgenic a-catenin-GFP protein, during morphological differentiation of adult wing cells in Drosophila. At the time of metamorphosis, wild-type cells are progressively transformed from a columnar hexagonal shape to a flattened shape, with a characteristic starry contour of cells. In the absence of the miniature ZP-domain gene, this remodeling of the shape of wing cells is prevented, with mutant cells displaying undifferentiated hexagonal contour (pictures by He´le`ne Chanut-Delalande).

differentiation of various embryonic and adult epithelial structures (Tables 1 and 2), including respiratory and epidermal derivatives [18,29,38–41]. Members of the ZP-domain family of proteins also are required for proper differentiation and function of sensory neurons, a feature conserved across considerable evolutionary distances. NompA accumulates in the extracellular space of the so-called dendritic cap of mechanosensory receptors and is required for sound perception in adult flies (Tables 1 and 2) [42]. Recently, a random genetic screen in nematodes identified the ZP-domain protein DYF-7 as being required for dendrite and glial process extension in the amphids [30], a pair of sense organs that mediate response to mechanical or chemical stimuli and regulate mating [43]. Therefore, ZP-domain proteins are expressed in many cell types during development and play important roles in

the terminal differentiation and function of neuro-epithelial cells and tissues. ZP-domain proteins in human diseases Independent evidence for the role of ZP-domain proteins in cell differentiation comes from identification of mutations in genes encoding ZP-domain proteins as the cause of human genetic diseases. As in invertebrates, ZP-domain proteins are implicated in the function of specific neural cells in mammals. The a- and b-tectorins are present in the tectorial membrane, an apical gel matrix necessary for sound transmission to neural cells in the cochlea [44,45] (Tables 1 and 2). Mutations in tectorins lead to the absence or improper formation of this ECM, causing deafness in mice and humans [44,46,47]. The gene encoding hensin produces alternatively spliced isoforms [5] having different names, including DMBT1 (deleted in malignant brain

Table 2. Main functions attributed to ZP-domain proteins

Fertilization Mechanotransduction Molting Remodeling of cell and exoskeleton shape Tubule morphogenesis Cellular junctions Cell adhesion

Functions ZP-domain proteins Mammals Fly ZP1, ZP2, ZP3, (ZP4) Tectorins NompA Hensin

Dusky-like, Miniature, Trynity, Zye

Endoglin, Uromodulin Hensin

Piopio, Dumpy Piopio, Dumpy, Trynity, Zye Papillote, Dumpy

Worm

Fish VEa, VEb, VEg

DYF-7 NOAH-1 and 2 RAM-5, CUT-1, CUT-3, CUT-5, CUT-4, CUT-6 LET-653

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Review tumors) [48–50], and some of them are specific to sensory organs, such as the vomeronasal organ and von Ebner’s gland [51] (Table 1). The widespread deletion of hensin/ DMBT1 in certain tumors, or its decreased expression in others, suggests that hensin/DMBT1 act as a tumor suppressor [52]. Uromodulin is specifically expressed in cells of the thick ascending limb of the Henle’s loop in the kidney. This protein, once targeted to the apical membrane, is released into the tubular lumen [53] where it polymerizes into a gellike structure that modulates salt transport and urine concentration [28]. In humans, uromodulin mutations induce renal diseases (Tables 1 and 2). Although uromodulin null mice exhibit no major defects during development, the absence of uromodulin leads to formation of urinary stones and nephrocalcinosis [54] and to reduced defense against microbial infections [55]. The ZP-domain protein endoglin/C105 is predominantly expressed in endothelial cells [56] where it acts as a modulator of TGFb signaling [57]. Mice lacking endoglin display cardiovascular lesions and patients heterozygous for endoglin mutations exhibit arteriovenous malformations, suggesting that endoglin controls multiple aspects of blood vessel formation [58–60] (Tables 1 and 2). Aside from its proangiogenic role, endoglin has also been linked to malignant progression in mediating epithelial mesenchymal transition [61]. These data strongly suggest that, in addition to developmental processes, ZP-domain proteins control important aspects of human physiology. However, experimental manipulations of model organisms are required to unravel the molecular mechanisms of ZP-domain protein functions. ZP-domain proteins and cell shape control The precise mechanisms underlying functions of ZPdomain proteins in epithelial differentiation have remained elusive. However, recent work has shed new light on their mode of action in remodeling cell shape. First insights into the role of ZP-domain proteins in the control of cell shape came from studies by Al-Awqati who examined the terminal differentiation of kidney cells. The cortical collecting duct of the kidney comprises two morphologically distinct cell types, referred to as a- and b- intercalated cells. Whereas a-cells exhibit a highly differentiated morphology, with a columnar shape and apical microvilli, b-cells display a poorly differentiated flat shape [62]. Ex vivo assays, in which the switch from a- to bphenotype can be controlled experimentally, indicate that the ZP-domain protein hensin is a key player in causing changes in the morphology of these cells (Figure 2B). Although hensin monomers are inactive, formation of higher-ordered hensin polymers induces the conversion of b- into a-type cells [62]. Diverse factors regulate hensin activation; a prolyl cis-trans isomerase promotes formation of hensin tetramers [63,64] and galectin 3 stimulates formation of larger hensin fibers [65]. In addition, integrin activation also plays a role in the control of hensin polymerization [66], but how this impacts on the polarized secretion of hensin and the subsequent changes in intercalated cell shape remains unclear. 528

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Several outcomes from studies in Drosophila further emphasize the role of ZP-domain proteins in cell shape remodeling during development. The formation of fine unicellular tubes at the distal tips of tracheae results from cellular rearrangements of larger multicellular tubes [39,67]. Jazwinska and colleagues showed that Pio and Dumpy (Dp), two ZP-domain proteins accumulated in the lumen of tracheal cells, play a key role in the regulation of tube morphogenesis [39]. Interestingly, let-653 in worms, that encodes a ZP-domain protein possessing two PAN domains, is required for proper apical differentiation of the excretory cell [68]. This cell is the largest cell in nematodes and organizes four long canals, corresponding also to unicellular tubules [69]. Mutations in let-653 result in extremely large canal lumena, which swell into a series of fluid-filled cysts [68,70]. Two genes in Drosophila, dusky (dy) and miniature (m), encoding ZP-domain proteins, were initially identified from their similar mutant phenotypes, causing a strong reduction in the wing size of adult flies [40]. During metamorphosis wing cells undergo a profound change in morphology, passing from columnar cells with a regular hexagonal arrangement to flattened cells displaying a characteristic starry contour (Figure 2D). This change in cell morphology is accompanied by extensive cytoskeletal modifications and leads to a large increase in the apical surface of cells. Miniature protein is incorporated into the aECM of wing cells and its inactivation causes prominent defects in cuticle layers. Cells lacking m are no longer able to flatten properly, explaining the general reduction in wing size observed in mutants [40]. The ZP-domain protein Dp, and Papillote (Pot), a protein containing only a ZP-N domain, are both required for adhesion to the aECM in wing cells [29]. Consequently, the inactivation of dp or pot causes the formation of wing blisters, a phenotype similar to that observed following alteration of the integrin pathway, which impairs cell adhesion via their basal surface [71]. A subpopulation of epidermal cells of the Drosophila embryo builds robust apical extensions, called denticles, which result from a localized bundling of actin filaments during terminal differentiation [72]. It has been well established that the stereotyped pattern of denticles is governed by specific expression of the transcription factor Shavenbaby (Svb) [73–75]. Identification of its target genes has revealed, in addition to actin regulators, the involvement of 8 ZP-domain proteins as required effectors of localized changes in the shape of epidermal cells [18,38]. Parallel analysis of these 8 ZP-domain genes shows that they each contribute to a specific aspect of denticle formation, with the individual inactivation of a given gene producing a characteristic defect in the shape of denticles (Figure 2C). Each protein accumulates in and organizes aECM layers of a restricted sub-apical region of the growing extension (Figure 3). Indeed, ultrastructural analyses reveal that the lack of each ZP-domain protein induces local defects in membrane/aECM interactions, at the precise location where the protein normally incorporates [18]. Together with their adjacent distribution along the epidermal extensions, the general properties of ZP-domain proteins to establish polymers suggest that these eight proteins

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Figure 3. Influence of ZP-domain proteins on apical cell junctions during Drosophila embryogenesis. (a) Role of Pio in the intercalation of tracheal cells, as observed by live imaging of a-catenin-GFP (pictures from [39]). The formation of thin tracheal tubes results from the dynamic remodeling of cell junctions, passing from lateral intercellular Adherens Junctions (iAJ) that connect two side-by-side tracheal cells (left), to autocellular Adherens Junctions (aAJ) (right). In wild type, tracheal cells that form unicellular tubes stay eventually connected with their neighbors only at the dorsal and ventral tips, leading to intercellular junction with lariat appearance that underline the lumen. Pio that accumulates in the lumen is required for this remodeling of junctional complexes. In the absence of Pio (or Dp, a second ZP-domain protein involved in this process), the conversion of intercellular to autocellular connectivity does not stop at the tip of cells, which detach from each other leading to tube disruption. Drawings at the right schematize defects in cell junctions and lumen formation that occur in the absence of Pio. (b) Role of Trynity (Tyn) during the morphological differentiation of epidermal cells, leading to denticle formation in a subset of those (purple lines). Cell junctions (observed from the apex) outline the apical constriction of denticle cells, compared with adjacent smooth cells that display a larger apical surface. In addition, denticle cells accumulate apical determinants (such as those of the Marginal Zone, MZ) and components of Adherens Junctions (AJ), as illustrated by a stronger DE cadherin staining than in smooth cells. Several ZP-domain proteins (Dyl, M, Tyn and Zye) are specifically distributed within the apical compartment, along the cytoplasmic extension underlying nascent denticles. Try and Zye localize to the base of each extension, down to apical junctions. The lack of Tyn (or Zye) that alters the shape of denticles prevents this reinforcement of apical cell junctions and leads to an enlarged apical cell contour (from [18]).

organize an ordered extracellular scaffold that finely sculpts the shape of apical cell extensions. These studies provide functional evidence that ZPdomain proteins contribute to the regulation of cell morphology, from its general organization to local inputs for formation of highly specialized apical extensions. ZP-domain proteins and the polarized organization of epithelial cells Reorganization of cell junctions is crucial for tissue morphogenesis during embryonic development, for example during germ band extension and mesoderm invagination in flies [76]. Such a remodeling in cell junctions is also likely to occur when Drosophila epidermal cells stretch along the dorsal region of the embryo to encompass the extra-embryonic amnioserosa [77–79]. In addition, modification of junctional complexes also takes place during the morphological differentiation of epidermal cells, specifically in those undergoing production of denticles [80]. When compared with their neighboring smooth cells, denticle cells accumulate high levels of the various components of apical junctions (Figure 3B), including DE-cadherin and Crumbs [18,80,81]. Interestingly, the distribution of two epidermal ZP-domain proteins, Trynity and Zye that are specific for denticle cells, normally extends to junctional complexes, whose characteristic accumulation is lost when one or both of the ZP-domain genes is inactivated [18]. These data suggest that ZP-domain proteins mediate their

role in remodeling of the apical compartment, at least in part, through maintenance of high levels of junctional complexes and other apical determinants in epidermal cells [18]. Interestingly, the ZP-domain proteins Pio and Dp also influence remodeling of junctional complexes during formation of thin tracheal branches [39], regulating the dynamic exchange from intercellular junctions to auto cellular junctions (Figure 3A). Recently it has been demonstrated that proper delivery of Pio in the tracheal lumen involves polarized trafficking machinery, with gCOP [82], Diaphanous (a Formin actin-nucleating factor) and the MyosinV motor [83]. Therefore, whereas the subcellular distribution of ZP-domain proteins relies on apical determinants, ZP-domain proteins might regulate in turn formation of cellular junctions. This interpretation is supported by the observation that hensin polymers influence cell polarity and promote the formation of junctional complexes [62,84]. It is probable that the effects of some ZP-domain proteins on cellular junctions reflect an indirect mechanism, for example as a result of physical constraints imposed by the polymerization of the extracellular region of ZP-domain proteins and transmitted to the underlying membrane compartment. However, a two-hybrid screen recently identified proto-cadherins as direct partners of ZP3 [85], opening the possibility of a more direct impact of ZP-domain proteins on adherence molecules. In a similar vein, indirect evidence suggests that ZP-domain proteins might functionally interact with components of the actin or 529

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Box 1. Outstanding questions The tissue-specific expression of ZP-domain genes is required at several steps of development and alteration of their transcriptional control has been linked in some cases with severe pathologies, including cancers. Little is known, however, about the molecular mechanisms that control the transcription of ZP-domain genes; both cis-regulatory regions and associated trans-regulators remain to be identified. To be active, ZP-domain proteins require a complex series of post-translational modifications, from the proper formation of intramolecular disulfides to the covalent addition of various sugar molecules. How the different steps of ZP-domain protein maturation are regulated and identification of the enzymatic activities responsible for these post-translational modifications represent challenging issues, illustrated in Figure I. In addition, the function of ZPdomain protein often relies on functional interactions that occur between different members of the ZP-domain family of proteins, coexpressed in the same cells. The observation that several ZP-domain proteins are targeted to distinct subapical regions of the plasma membrane provide evidence that the routing of ZP-domain proteins to specific membrane subdomains probably involves highly regulated trafficking. Genetic approaches in flies and worms open promising ways to explore the molecular mechanisms regulating ZP-domain protein delivery, a matter that can also be linked to their intramolecular association. Indeed, there are many examples showing the functional importance of the hetero- and/or homo-polymerization of ZP-domain proteins. Understanding the recognition code responsible for the regulated polymerization of ZP-domain proteins is a crucial aspect of future work. Finally, it is well documented that ZP-domain proteins are subjected to proteolytic cleavage, leading to the release of their extracellular portion (including the ZP-domain) from the membrane anchored C-terminal tail. Both the functional importance of this cleavage and the mechanisms involved will require further investigation.

[(Box_1)TD$FIG]

microtubule cytoskeleton [29,40,86], although the basis of these interactions is unclear. Future directions A common theme for the roles of ZP-domain proteins in morphological differentiation is the combined activity of several members of this protein family and, at least in some cases, this requires their delivery to distinct membrane (sub) compartments. At the present time the trafficking machinery responsible for maturation and targeting of ZP-domain proteins is poorly understood (Box 1). Nevertheless, recent findings that certain ZPdomain proteins are targeted to and define sub-regions within the apical compartment [18] offer new means to explore the mechanisms involved. Genetic screening in model organisms could provide efficient means to identify factors involved in regulating specific targeting of ZPdomain proteins, vesicle trafficking, and protein secretion [82,83]. A second crucial issue is to elucidate the polymerization properties of ZP-domain proteins and their possible modulation by other motifs or domains. Development of suitable reconstituted assays using cultured cells, such as those recently initiated by Schaeffer et al. on uromodulin [28], represents an important advance. Finally, unraveling mechanisms involved in post-translational modifications of ZP-domain proteins, including regulation of their cleavage [29,30], would benefit from further structural studies and from in vivo analyses. 530

Figure I. Successive steps involved in the functional maturation of ZP-domain proteins.

Collectively, recent progress made on biochemical and structural aspects of ZP-domain proteins and in establishing attractive genetic systems to study ZP-domain proteins will undoubtedly lead to a deeper understanding of how these proteins regulate various aspects of cell differentiation and remodeling of cell shape. Acknowledgements This work was supported by grants from the Association pour la Recherche contre le Cancer (no. 3832, no. 1111 and a fellowship to I.F.), Fondation pour la Recherche Me´dicale (e´quipe 2005), and ANR Blanc Netoshape. The Wassarman laboratory was supported by the National Institute of Health (NICHD), most recently by grant HD-35105. We thank P. Ferrer for critical reading of the manuscript. The authors have declared no conflict of interest.

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