Positional cloning of heart and soul reveals multiple roles for PKC lambda in zebrafish organogenesis

BACKGROUND: The Par-3/Par-6/aPKC complex is a key regulator of cell polarity in a number of systems. In Drosophila, this complex acts at the zonula adherens (adherens junctions) to establish epithelial polarity and helps to orient the mitotic spindle during asymmetric neuroblast divisions. In MDCKII cells, this complex localizes to the zonula occludens (tight junctions) and appears to regulate epithelial polarity. However, the in vivo role of this complex during vertebrate embryogenesis is not known, due to the lack of relevant mutations. RESULTS: We have positionally cloned the zebrafish heart and soul (has) mutation, which affects the morphogenesis of several embryonic tissues, and show that it encodes atypical protein kinase C lambda (aPKC lambda). We find that loss of aPKC lambda affects the formation and maintenance of the zonula adherens in the polarized epithelia of the retina, neural tube, and digestive tract, leading to novel phenotypes, such as the formation of multiple lumens in the developing intestine. In addition, has mutants display defects in gut looping and endodermal organ morphogenesis that appear to be independent of the defects in epithelial polarity. Finally, we show that loss of aPKC lambda leads to defects in spindle orientation during progenitor cell divisions in the neural retina. CONCLUSIONS: Our results show that aPKC lambda is required for the formation and maintenance of the zonula adherens during early epithelial development in vertebrates and demonstrate a previously undescribed yet critical role for this protein in organ morphogenesis. Furthermore, our studies identify the first genetic locus regulating the orientation of cell division in vertebrates.     

Sticky business: orchestrating cellular signals at adherens junctions

Cohesive sheets of epithelial cells are a fundamental feature of multicellular organisms and are largely a product of the varied functions of adherens junctions. These junctions and their cytoskeletal associations contribute heavily to the distinct shapes, polarity, spatially oriented mitotic spindle planes, and cellular movements of developing tissues. Deciphering the underlying mechanisms that govern these conserved cellular rearrangements is a prerequisite to understanding vertebrate morphogenesis.     

FRAP Analysis Reveals Stabilization of Adhesion Structures in the Epidermis Compared to Cultured Keratinocytes

Proper development and tissue maintenance requires cell-cell adhesion structures, which serve diverse and crucial roles in tissue morphogenesis. Epithelial tissues have three main types of cell-cell junctions: tight junctions, which play a major role in barrier formation, and adherens junctions and desmosomes, which provide mechanical stability and organize the underlying cytoskeleton. Our current understanding of adhesion function is hindered by a lack of tools and methods to image junctions in mammals. To better understand the dynamics of adhesion in tissues we have created a knock-in ZO-1-GFP mouse and a BAC-transgenic mouse expressing desmoplakin I-GFP. We performed fluorescence recovery after photobleaching (FRAP) experiments to quantify the turnover rates of the tight junction protein ZO-1, the adherens junction protein E-cadherin, and the desmosomal protein desmoplakin in the epidermis. Proteins at each type of junction are remarkably stable in the epidermis, in contrast to the high observed mobility of E-cadherin and ZO-1 at adherens junctions and tight junctions, respectively, in cultured cells. Our data demonstrate that there are additional mechanisms for stabilizing junctions in tissues that are not modeled by cell culture.     

Abelson kinase regulates epithelial morphogenesis in Drosophila.

Activation of the nonreceptor tyrosine kinase Abelson (Abl) contributes to the development of leukemia, but the complex roles of Abl in normal development are not fully understood. Drosophila Abl links neural axon guidance receptors to the cytoskeleton. Here we report a novel role for Drosophila Abl in epithelial cells, where it is critical for morphogenesis. Embryos completely lacking both maternal and zygotic Abl die with defects in several morphogenetic processes requiring cell shape changes and cell migration. We describe the cellular defects that underlie these problems, focusing on dorsal closure as an example. Further, we show that the Abl target Enabled (Ena), a modulator of actin dynamics, is involved with Abl in morphogenesis. We find that Ena localizes to adherens junctions of most epithelial cells, and that it genetically interacts with the adherens junction protein Armadillo (Arm) during morphogenesis. The defects of abl mutants are strongly enhanced by heterozygosity for shotgun, which encodes DE-cadherin. Finally, loss of Abl reduces Arm and alpha-catenin accumulation in adherens junctions, while having little or no effect on other components of the cytoskeleton or cell polarity machinery. We discuss possible models for Abl function during epithelial morphogenesis in light of these data.     

Adherens junctions: new insight into assembly,modulation and function

Adherens junctions play pivotal roles in cell and tissue organization and patterning by mediating cell adhesion and cell signaling. These junctions consist of large multiprotein complexes that join the actin cytoskeleton to the plasma membrane to form adhesive contacts between cells or between cells and extracellular matrix. The best-known adherens junction is the zonula adherens (ZA) that forms a belt surrounding the apical pole of epithelial cells. Recent studies in Drosophila have further illuminated the structure of adherens junctions. Scaffolding proteins encoded by the stardust gene are novel components of the Crumbs complex, which plays a critical role in ZA assembly.1-3 The small GTPase Rap1 controls the symmetric re-assembly of the ZA after cell division.4 Finally, the asymmetric distribution of adherens junction material regulates spindle orientation during asymmetric cell division in the sensory organ lineage.     

E-N-cadherin heterodimers define novel adherens junctions connecting endoderm-derived cells

Intercellular junctions play a pivotal role in tissue development and function and also in tumorigenesis. In epithelial cells, decrease or loss of E-cadherin, the hallmark molecule of adherens junctions (AJs), and increase of N-cadherin are widely thought to promote carcinoma progression and metastasis. In this paper, we show that this "cadherin switch" hypothesis does not hold for diverse endoderm-derived cells and cells of tumors derived from them. We show that the cadherins in a major portion of AJs in these cells can be chemically cross-linked in E-N heterodimers. We also show that cells possessing E-N heterodimer AJs can form semistable hemihomotypic AJs with purely N-cadherin-based AJs of mesenchymally derived cells, including stroma cells. We conclude that these heterodimers are the major AJ constituents of several endoderm-derived tissues and tumors and that the prevailing concept of antagonistic roles of these two cadherins in developmental and tumor biology has to be reconsidered.     

Syntaxin 16 Regulates Lumen Formation during Epithelial Morphogenesis

The formation and maintenance of cell-cell junctions, both under physiological and pathological conditions, requires the targeting and trafficking of junctional proteins. Proteins of the syntaxin (Stx)-family localize to a variety of subcellular membranes and contribute to intracellular transport of cargo by regulating vesicle fusion events at these sites. Unlike plasma membrane localized Stxs, the roles of endosome- and Golgi-localized stx proteins in epithelial morphogenesis are less understood. Here we show that Stx16– an endosome- and Golgi-localized target-membrane soluble N-ethylmaleimide attachment protein receptor (t-SNARE) that plays a role in membrane trafficking between these compartments – is essential for lumen development. In cultured Madin Darby Canine Kidney (MDCK) cells, Stx16 was selectively upregulated as sparsely plated cells attained confluency. Stx16-depleted confluent monolayers consistently showed lower transepithelial resistance than control monolayers, and failed to maintain endogenous and ectopically expressed E-cadherin at the adherens junctions due to decreased recycling. We further found that whereas cysts formed by MDCK cells cultured in Matrigel have a single hollow lumen, those formed by stx16-depleted counterparts had multiple lumens, due to abnormal orientiation of the mitotic spindle. Finally, a similar role for stx16 function in vivo is indicated by our analysis of pronephric-duct development in zebrafish expressing the claudinB:lynGFP transgene; lack of stx16 function in this structure (in stx16-morphant embryos) led to the development of enlarged, torturous pronephric ducts with more than one lumen. Taken together, our in vitro and in vivo studies establish a role for Stx16 in maintaining the integrity of cell-cell junctions, and thereby in morphogenesis of the kidney epithelial lumen.     

E-cadherin is required for intestinal morphogenesis in the mouse

E-cadherin, the primary epithelial adherens junction protein, has been implicated as playing a critical role in nucleating formation of adherens junctions, tight junctions, and desmosomes. In addition to its role in maintaining structural tissue integrity, E-cadherin has also been suggested as an important modulator of cell signaling via interactions with its cytoplasmic binding partners, catenins, as well as with growth factor receptors. Therefore, we proposed that loss of E-cadherin from the developing mouse intestinal epithelium would disrupt intestinal epithelial morphogenesis and function. To test this hypothesis, we used a conditional knockout approach to eliminate E-cadherin specifically in the intestinal epithelium during embryonic development. We found that E-cadherin conditional knockout mice failed to survive, dying within the first 24 hours of birth. Examination of intestinal architecture at E18.5 demonstrated severe disruption to intestinal morphogenesis in animals lacking E-cadherin in the epithelium of the small intestine. We observed changes in epithelial cell shape as well as in the morphology of villi. Although junctional complexes were evident, junctions were abnormal, and barrier function was compromised in E-cadherin mutant intestine. We also identified changes in the epithelial cell populations present in E-cadherin conditional knockout animals. The number of proliferating cells was increased, whereas the number of enterocytes was decreased. Although Wnt/β-catenin target mRNAs were more abundant in mutants compared with controls, the amount of nuclear activated β-catenin protein was dramatically lower in mutants compared with controls. In summary, our data demonstrate that E-cadherin is essential for intestinal epithelial morphogenesis and homeostasis during embryonic development.     

Control of polarized cell morphology and motility by adherens junctions

Cell-cell interactions play a key role in tissue homeostasis. Intercellular adhesions share the complex task of establishing and maintaining tissue architecture while allowing tissue growth, renewal and repair. In particular, adherens junctions (AJs) have been implicated in the formation of diverse tissues and organs like epitheliums, blood vessels or the central nervous system. At the cellular level, AJs are well known for their essential role in epithelial cell differentiation and baso-apical polarity. They also contribute to the control of cell polarity to promote neuronal morphogenesis, growth cone guidance and directed migration in a variety of cell types during embryonic development. AJs based on classical cadherin- and nectin-mediated cell-cell interactions control local membrane dynamics to polarize cell morphology and motility at the single cell level and to coordinate cell shape changes and motile behaviour at the tissue level. I review here the molecular mechanisms allowing control of polarized cell morphology and motility by AJs.     

HAS3-induced accumulation of hyaluronan in 3D MDCK cultures results in mitotic spindle misorientation and disturbed organization of epithelium

The amount of hyaluronan (HA) is low in simple epithelia under normal conditions, but during tumorigenesis, trauma or inflammation HA is increased on the epithelial cells and surrounding stroma. Excessive HA in epithelia is suggested to interfere with cell–cell adhesions, resulting in disruption of the epithelial barrier function. In addition, stimulated HA synthesis has been correlated with epithelial-to-mesenchymal transition and invasion of cancer cells. However, the effects of HA overload on normal epithelial morphogenesis have not been characterized in detail. Madin-Darby canine kidney (MDCK) cells form polarized epithelial cysts, when grown in a 3-dimensional (3D) matrix. These cells were used to investigate whether stimulated HA synthesis, induced by stable overexpression of GFP-HAS3, influences cell polarization and epithelial morphogenesis. GFP-HAS3 expression in polarized MDCK cells resulted in active HA secretion at apical and basolateral membrane domains. HA-deposits interfered with the formation of cell–cell junctions, resulting in impaired barrier function. In 3D cyst cultures, HA accumulated into apical lumina and was also secreted from the basal side. The HAS3-expressing cysts failed to form a single lumen and instead displayed multiple small lumina. This phenotype was correlated with aberrant mitotic spindle orientation in dividing cells. The results of this study indicate that excess pericellular HA disturbs the normal cell–cell and cell–ECM interactions in simple epithelia, leading to aberrant epithelial morphogenesis. The morphological abnormalities observed in 3D epithelial cultures upon stimulated HAS3 expression may be related to premalignant changes, including intraluminal invasion and deregulated epithelialization, probably mediated by the mitotic spindle orientation defects.     

Epithelial polarity and morphogenesis

The adult form of a multicellular organism is shaped by a series of morphogenetic processes that organise the body into tissues and organs. Most of these events involve the deformation of sheets of epithelial cells that are highly polarised along their apical-basal axes and attached to each other by lateral junctions. Here we discuss the role played by modifications in the apical-basal polarity system in driving morphogenesis, with an emphasis on well-characterised events during Drosophila development. Changing the activity of polarity factors can alter the relative sizes of the apical, lateral and basal domains. This can drive transitions between cuboidal, columnar and squamous epithelial morphologies, to increase or decrease the surface area of an epithelial sheet. These changes can also cause epithelial cells to become wedge-shaped, which can drive tissue bending and invagination. In addition, it has recently emerged that the activity of apical-basal polarity factors can also be modulated in a planar polarised manner. By affecting the contractility of the actomyosin cytoskeleton and the stability of adherens junctions, changes within the plane of the epithelium can cause cell rearrangements that contribute to convergence and extension movements, boundary formation and cell alignment.     

The NF2 tumor suppressor, Merlin, regulates epidermal development through the establishment of a junctional polarity complex

The neurofibromatosis type 2 (NF2) tumor suppressor, Merlin, is a FERM (Four point one, Ezrin, Radixin, Moesin) domain-containing protein whose loss results in defective morphogenesis and tumorigenesis in multiple tissues. Like the closely related ERM proteins (Ezrin, Radixin, and Moesin), Merlin may organize the plasma membrane by assembling membrane protein complexes and linking them to the cortical actin cytoskeleton. We previously found that Merlin is a critical mediator of contact-dependent inhibition of proliferation and is required for the establishment of stable adherens junctions (AJs) in cultured cells. Here, we delineate the molecular function of Merlin in AJ establishment in epidermal keratinocytes in?vitro and confirm that a role in AJ establishment is an essential function of Merlin in?vivo. Our studies reveal that Merlin can associate directly with α-catenin and link it to Par3, thereby providing an essential link between the AJ and the Par3 polarity complex during junctional maturation.     

Oriented cell division in vertebrate embryogenesis

Tissue morphogenesis depends on the spatial arrangement of cells during development. A number of mechanisms have been described to contribute to the final shape of a tissue or organ, ranging from cell intercalation to the response of cells to chemotactic cues. One such mechanism is oriented cell division. Oriented cell division is determined by the position of the mitotic spindle. Indeed, there is increasing evidence implicating spindle misorientation in tissue and organ misshaping, which underlies disease conditions such as tumorigenesis or polycystic kidneys. Here we review recent studies addressing how the direction of tissue growth is determined by the orientation of cell division and how both extrinsic and intrinsic cues control the position of the mitotic spindle.     

Distinct roles of cadherin-6 and E-cadherin in tubulogenesis and lumen formation

Classic cadherins are important regulators of tissue morphogenesis. The predominant cadherin in epithelial cells, E-cadherin, has been extensively studied because of its critical role in normal epithelial development and carcinogenesis. Epithelial cells may also coexpress other cadherins, but their roles are less clear. The Madin Darby canine kidney (MDCK) cell line has been a popular mammalian model to investigate the role of E-cadherin in epithelial polarization and tubulogenesis. However, MDCK cells also express relatively high levels of cadherin-6, and it is unclear whether the functions of this cadherin are redundant to those of E-cadherin. We investigate the specific roles of both cadherins using a knockdown approach. Although we find that both cadherins are able to form adherens junctions at the basolateral surface, we show that they have specific and mutually exclusive roles in epithelial morphogenesis. Specifically, we find that cadherin-6 functions as an inhibitor of tubulogenesis, whereas E-cadherin is required for lumen formation. Ablation of cadherin-6 leads to the spontaneous formation of tubules, which depends on increased phosphoinositide 3-kinase (PI3K) activity. In contrast, loss of E-cadherin inhibits lumen formation by a mechanism independent of PI3K.     

Formin-mediated actin polymerization at cell–cell junctions stabilizes E-cadherin and maintains monolayer integrity during wound repair

Cadherin-mediated cell–cell adhesion is required for epithelial tissue integrity in homeostasis, during development, and in tissue repair. E-cadherin stability depends on F-actin, but the mechanisms regulating actin polymerization at cell–cell junctions remain poorly understood. Here we investigated a role for formin-mediated actin polymerization at cell–cell junctions. We identify mDia1 and Fmnl3 as major factors enhancing actin polymerization and stabilizing E-cadherin at epithelial junctions. Fmnl3 localizes to adherens junctions downstream of Src and Cdc42 and its depletion leads to a reduction in F-actin and E-cadherin at junctions and a weakening of cell–cell adhesion. Of importance, Fmnl3 expression is up-regulated and junctional localization increases during collective cell migration. Depletion of Fmnl3 or mDia1 in migrating monolayers results in dissociation of leader cells and impaired wound repair. In summary, our results show that formin activity at epithelial cell–cell junctions is important for adhesion and the maintenance of epithelial cohesion during dynamic processes, such as wound repair.     

The Caenorhabditis elegans p120 catenin homologue,JAC-1, modulates cadherin-catenin function during epidermal morphogenesis

The cadherin-catenin complex is essential for tissue morphogenesis during animal development. In cultured mammalian cells, p120 catenin (p120ctn) is an important regulator of cadherin-catenin complex function. However, information on the role of p120ctn family members in cadherin-dependent events in vivo is limited. We have examined the role of the single Caenorhabditis elegans p120ctn homologue JAC-1 (juxtamembrane domain [JMD]-associated catenin) during epidermal morphogenesis. Similar to other p120ctn family members, JAC-1 binds the JMD of the classical cadherin HMR-1, and GFP-tagged JAC-1 localizes to adherens junctions in an HMR-1-dependent manner. Surprisingly, depleting JAC-1 expression using RNA interference (RNAi) does not result in any obvious defects in embryonic or postembryonic development. However, jac-1(RNAi) does increase the severity and penetrance of morphogenetic defects caused by a hypomorphic mutation in the hmp-1/alpha-catenin gene. In these hmp-1 mutants, jac-1 depletion causes failure of the embryo to elongate into a worm-like shape, a process that involves contraction of the epidermis. Associated with failed elongation is the detachment of actin bundles from epidermal adherens junctions and failure to maintain cadherin in adherens junctions. These results suggest that JAC-1 acts as a positive modulator of cadherin function in C. elegans.     

Cadherin Adhesion Receptors Orient the Mitotic Spindle during Symmetric Cell Division in Mammalian Epithelia

Oriented cell division is a fundamental determinant of tissue organization. Simple epithelia divide symmetrically in the plane of the monolayer to preserve organ structure during epithelial morphogenesis and tissue turnover. For this to occur, mitotic spindles must be stringently oriented in the Z-axis, thereby establishing the perpendicular division plane between daughter cells. Spatial cues are thought to play important roles in spindle orientation, notably during asymmetric cell division. The molecular nature of the cortical cues that guide the spindle during symmetric cell division, however, is poorly understood. Here we show directly for the first time that cadherin adhesion receptors are required for planar spindle orientation in mammalian epithelia. Importantly, spindle orientation was disrupted without affecting tissue cohesion or epithelial polarity. This suggests that cadherin receptors can serve as cues for spindle orientation during symmetric cell division. We further show that disrupting cadherin function perturbed the cortical localization of APC, a microtubule-interacting protein that was required for planar spindle orientation. Together, these findings establish a novel morphogenetic function for cadherin adhesion receptors to guide spindle orientation during symmetric cell division.