shotgun

DEVELOPMENTAL BIOLOGY

Effects of Mutation or Deletion

Various mutant classes lack portions of the head cuticle, show defects in head involution, have holes in ventral cuticle or lack most or all of the head and ventral epidermis. Some show segmentation defects, and others have dominant negative effects. The appearance of holes in the ventral cuticle has to do with the presence of gaps left by delaminating neuroblasts. Notch overexpression, preventing neuroblast delamination, reverses this phenotype.

An intermediate mutant allele of armadillo was used to explore the requirement for ARM in adherens junction assembly, cell polarity and morphogenesis in Drosophila. Adherens junctions cannot assemble in the absence of ARM; this leads to dramatic defects in cell-cell adhesion. The epithelial cells of the embryo lose adhesion to one another, round up, and apparently become mesenchymal. In arm mutants, alpha-catenin no longer accumulates at the plasma membrane, but instead is found diffusely in the cytoplasm. Shotgun, the Drosophila E-cadherin, is normally tightly localized to the plasma membrane and enriched in adherens junctions, but in arm mutants, Shotgun accumulation at the plasma membrane is reduced. Much of the remaining Shotgun accumulates within cells, presumably in the ER, Golgi, or endosomes. This may be a result of endocytosis. Mutant cells also lose their normal cell polarity. These disruptions to the integrity of the epithelia constitute a block to the appropriate morphogenetic movements of gastrulation. There is little or no germ band extention, and the ventral furrow and posterior midgut fail to invaginate normally. Reducing Shotgun levels suppresses the armadillo segment polarity phenotype. It has been suggested that this suppression reflects the fact that Armadillo's roles in adherens junctions and Wingless signaling are separable, and that under conditions where ARM is limiting, the reduction in the number of junctional complexes frees up some of the wild-type ARM, allowing it to function in the Wingless signaling (Cox, 1996).

Cadherins are involved in a variety of morphogenetic movements during animal development. However, it has been difficult to pinpoint the precise function of cadherins in morphogenetic processes due to the multifunctional nature of cadherin requirement. The data presented in this study indicate that homophilic adhesion promoted by Drosophila E-cadherin (DE-cadherin) mediates two cell migration events during Drosophila oogenesis. In Drosophila follicles, two groups of follicle cells, the border cells and the centripetal cells migrate on the surface of germline cells. The border cells migrate as an epithelial patch in which two centrally located cells retain epithelial polarity and peripheral cells are partially depolarized. Both follicle cells and germline cells express DE-cadherin, and border cells and centripetal cells strongly upregulate the expression of DE-cadherin shortly before and during their migration. Removing DE-cadherin from either the follicle cells or the germline cells blocks migration of border cells and centripetal cells on the surface of germline cells. The function of DE-cadherin in border cells appears to be specific for migration as the formation of the border cell cluster and the adhesion between border cells are not disrupted in the absence of DE-cadherin. The speed of migration depends on the level of DE-cadherin expression, as border cells migrate more slowly when DE-cadherin activity is reduced. Finally, it is shown that the upregulation of DE-cadherin expression in border cells depends on the activity of the Drosophila C/EBP transcription factor that is essential for border cell migration (Niewiadomska, 1999).

Heart development in the Drosophila embryo starts with the specification of cardiac precursors from the dorsal edge of the mesoderm and continues through signaling from the epidermis. Cardioblasts then become aligned in a single row of cells that migrate dorsally. After contacting their contralateral counterparts, cardioblasts undergo a cytoskeletal rearrangement and form a lumen. Its simple architecture and cellular composition makes the heart a good system to study mesodermal patterning, intergerm layer signaling, and the function of cell adhesion molecules (CAMs) during morphogenesis. A focus is placed on three adhesion molecules essential for heart development: faint sausage (fas), shotgun (E-cadherin), and Laminin A (Lam A). fas encodes an Ig-like CAM and is required for the correct number of cardioblasts to become specified, as well as proper alignment of cardioblasts. Those specified in mutants tend to migrate abnormally away from the midline. A similar qualitative phenotype can be achieved by reducing Notch function during cardiac development, suggesting that fas may play a permissive role during Notch/Delta-mediated cell-cell interactions among heart precursors. shg is expressed and required at a later stage than fas; in embryos lacking this gene, cardioblasts are specified normally and become aligned, but do not form a lumen. Additionally, cardioblasts of shg mutant embryos show a redistribution of phosphotyrosine as well as a loss of Armadillo from the membrane, indicating defects in cell polarity. Both Arm and phosphotyrosine are markers for the zonula adherens (ZA). Wild-type cardioblasts have a ZA that mediates the contact between neighboring cardioblasts of the same side of the embryo. The lateral segment of the AZ (between ipsilateral cardioblasts) is unaffected. Cells of opposite sides do not form a junctional complex with each other in shg mutants; they also do not bend into the crescent shape typical of wild-type cardioblasts. The shg phenotype can be phenocopied by applying EGTA or cytochalasin D, supporting the view that Ca2+-dependent adhesion and the actin cytoskeleton are instrumental for heart lumen formation. As opposed to cell-cell adhesion, cell-substrate adhesion mechanisms are not required for heart morphogenesis, but only for maintenance of the differentiated heart. Embryos lacking the LamA gene initially develop a normal heart, but show twists and breaks of cardioblasts at late embryonic stages. These findings are viewed in the light of recent results that elucidate the function of different adhesion systems in vertebrate heart development. The function of Shg during cardioblast lumen formation appears similar to the presumed role of VE-cadherin in vertebrate heart and capillary development. In the chick, VE-adherin expression increases in the endocardial primordium and N-cadherin is turned off (Haag, 1999).

Two viable fly stocks have been generated by altering the level of Armadillo available for signaling. Flies from one stock overexpress Armadillo (Arm^over) and, as a result, have increased vein material and bristles in the wings. Flies from the other stock have reduced cytoplasmic Armadillo following overexpression of the intracellular domain of DE-cadherin (Arm^under). These flies display a wing-notching phenotype typical of wingless mutations. Both misexpression phenotypes can be dominantly modified by removing one copy of genes known to encode members of the wingless pathway. This paper identifies and describes further mutations that dominantly modify the Armadillo misexpression phenotypes. These mutations are in genes encoding three different functions: establishment and maintenance of adherens junctions, cell cycle control, and Egfr signaling (Greaves, 1999).

Mutations in 17 genes (26 deficiencies) were characterized that interact with Arm^over and/or Arm^under. Interaction strength varies from deficiency to point mutation, suggesting that several genes in the original deficiencies could have contributed to, or modified, the interaction. Only for 7 of the 17 genes have interactions been identical between the point mutation and the corresponding starting deficiency. The 17 genes were sorted into four groups. Group 2 consists of genes required for cell adhesion: This group includes shotgun (which encodes DE-cadherin), as expected. Also uncovered were fat (ft) and dachsous (ds). These two genes encode nonclassical cadherin characterized by a huge extracellular domain containing up to 35 cadherin repeats and a bipartite Arm binding site. Interactions with these two mutants are similar to those observed with shotgun (DE-cadherin), the only difference being that ft interacts more weakly than shg with Arm^over. In addition to genes encoding cadherins (classical and nonclassical), interactions have been observed with some of the genes known to be essential (directly or indirectly) for the assembly or maintenance of adherens junctions—stardust (sdt), discs-large (dlg), and crumbs (crb). These interact in the same direction as shg; however, the suppression of Arm^under is always weaker and only dlg^M52 enhances Arm^over to the same extent as zw3^M11 (Greaves, 1999).

The interaction with shotgun (encoding DE-cadherin) itself is not very illuminating since it is expected that the phenotype caused by an excess of intracellular cadherin domain will be suppressed by decreasing endogenous cadherin levels. Still, this interaction shows that the level of overexpression afforded by the Gal4p system is within physiological levels. Interaction with fat and dachsous suggests that these two nonclassical cadherins interact (maybe directly) with Arm. Initial analysis of the intracellular domain of Fat and Dachsous fail to identify an Arm/ß-catenin binding site homologous to that found in E-cadherin. However, subsequent sequence examination suggests the existence of a bipartite site. Genetic interactions with fat and dachsous strongly suggest that this proposed site is functional, and thus removing one copy of the fat or dachsous gene would release additional Arm to the cytoplasm and make it available for use in Wg transduction. Interactions with fat and dachsous in the eye confirm the ability of these genes to modify cytoplasmic Arm levels. It also indicates that these genes are expressed in the eye and may be functional there (Greaves, 1999).

Cadherin-N (CadN) binds to Arm. Therefore the failure of CadN to interact in this screen suggests that CadN may not be expressed to significant levels in the posterior compartment of wing imaginal discs or in eye precursors. In contrast, crumbs (crb) and stardust (sdt) do interact. The proteins encoded by these genes are not thought to participate in junctional complexes per se. Rather, they control the biogenesis of the junctions. It is suggested that decreasing the activity of crb or sdt has a quantitative effect on the number or size of adherens junctions and this would lead to more Arm being released from the membrane and made available for Wg signaling (Greaves, 1999).

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. This study reports 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. The cellular defects are described that underlie these problems, focusing on dorsal closure as an example. Further, it is shown that the Abl target Enabled (Ena), a modulator of actin dynamics, is involved with Abl in morphogenesis. Ena localizes to adherens junctions of most epithelial cells, and 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. Possible models for Abl function during epithelial morphogenesis are discussed in light of these data (Grevengoed, 2001).

Several lines of evidence support the possibility that the morphogenetic defects of abl^MZ mutants result, at least in part, from Abl action at adherens junctions. (1) The effects on dorsal closure, germband retraction, and head involution are strongly enhanced by reducing the dose of DE-cadherin. (2) The defects in cell shape during dorsal closure resemble, in part, those of arm mutants. (3) The defects in morphogenesis are suppressed by mutations in ena, which is primarily found at adherens junctions. (4) A reduction in junctional Arm and alpha-catenin is seen in abl^MZ mutants. It is important to note, however, that any role for Abl at adherens junctions would be a modulatory one. It is not absolutely essential for adherens junction assembly or function. Of course, it remains possible that other tyrosine kinases may act redundantly with Abl. The relationship between the cadherin-catenin system, Abl, and Ena that may occur in epithelial cells could also exist in the CNS. Arm and DN-cadherin play roles in axon outgrowth in Drosophila, and in this role arm interacts genetically with abl (Grevengoed, 2001).

One target of Abl might be Ena, which could regulate actin dynamics in the actin belt underlying the adherens junction. Just as local modulation of actin dynamics likely regulates growth cone extension or stalling, the cell shape changes and cell migration characteristic of morphogenesis will require modulation of actin dynamics and junctional linkage. The idea that Ena may regulate cell-cell adhesion recently received strong support from work in cultured mammalian keratinocytes, where inhibiting Ena/VASP function prevented actin rearrangement upon cell-cell adhesion. This model is further supported by the demonstration that both Ab1 and Ena regulate actin polymerization at the adherens junctions of ovarian follicle cells in Drosophila (Grevengoed, 2001).

Generation of cell-fate diversity in Metazoan depends in part on asymmetric cell divisions in which cell-fate determinants are asymmetrically distributed in the mother cell and unequally partitioned between daughter cells. The polarization of the mother cell is a prerequisite to the unequal segregation of cell-fate determinants. In the Drosophila bristle lineage, two distinct mechanisms are known to define the axis of polarity of the pI and pIIb cells. Frizzled (Fz) signaling regulates the planar orientation of the pI division, while Inscuteable (Insc) directs the apical-basal polarity of the pIIb cell. The orientation of the asymmetric division of the pIIa cell is identical to the orientation of its mother cell, the pI cell, but, in contrast, is regulated by an unknown Insc- and Fz-independent mechanism. Drosophila E-Cadherin-Catenin (Shotgun-Armadillo) complexes are shown to localize at the cell contact between the two cells born from the asymmetric division of the pI cell. The mitotic spindle of the dividing pIIa cell rotates to line up with asymmetrically localized Shotgun-Armadillo complexes. While a complete loss of Shotgun function disrupts the apical-basal polarity of the epithelium, both a partial loss of Shotgun function and expression of a dominant-negative form of Shotgun affect the orientation of the pIIa division. Furthermore, expression of dominant-negative Shotgun also affects the position of Partner of Inscuteable (Pins) and Bazooka, two asymmetrically localized proteins known to regulate cell polarity. These results show that asymmetrically distributed Shotgun regulates the orientation of asymmetric cell division (Le Borgne, 2002).

Three distinct mechanisms regulate the stereotyped orientation of the first three asymmetric cell divisions in the seemingly simple lineage that generates the sense organs on the Drosophila notum. (1) In the pI cell, Fz signaling orients the mitotic spindle along the AP axis of the body, regulates the formation of the Dlg/Pins and Baz complexes at the anterior and posterior poles, respectively, and thereby directs the asymmetric localization of the Numb crescent to the anterior cortex. (2) By analogy to the neuroblasts, an apical Baz/Insc/Pins complex is thought to direct the apical-basal orientation of the pIIb division. This analogy is supported by the observation that Pins, Baz, and Insc colocalize at the apical cortex of the dividing pIIb cell. (3) The pIIa cell divides with the same orientation as its mother cell in a Fz- and Insc-independent manner. In the pIIa cell, a specific cortical domain formed at the region of cell-cell contact between the pIIb/pIIIb and pIIa cells appears to regulate the precise orientation of this division. Five lines of evidence support this last conclusion: (1) Shotgun (Shg), Arm, and alpha-Catenin-GFP localize asymmetrically in a cortical patch at the anterior pole of the dividing pIIa cell; (2) the mitotic spindle of the pIIa cell rotates to specifically line up with this cortical domain; (3) expression of a dominant-negative form of Shg perturbs both the formation of this cortical domain, the orientation of the pIIa division, and the precise positioning of Pins at the anterior lateral cortex; (4) loss of Shg activity in clones leads to defects in the orientation of the pIIa division; (5) Pins localizes opposite of Baz in the pIIa cell along a polarity axis defined by the patch of Shg, and dominant-negative Shg affects the orientation of these two domains relative to this patch. Noticeably, a strong loss of Shg function does not randomize the orientation of the mitotic spindle or of the Pins/Baz domains. Thus, one function of Shg in the pIIa cell is to ensure precision in the orientation of the polarity axis. Although loss of Fz activity randomizes the orientation of the pI cell, Shg appears to play a role formally similar to Fz in defining the polarity axis in the pIIa cell. This is the first evidence of a regulatory role of E-Cadherin in the orientation of asymmetric cell divisions (Le Borgne, 2002).

Time-lapse imaging results suggest that molecules localized at or near the anterior cortical patch capture the anterior centrosome, therefore leading to a rotation of the spindle. Centrosome capture is thought to depend on direct interactions between microtubule-bound proteins and specific cortical proteins. Because the anterior centrosome is located basal to the Shg-containing cortical domain, it is suggested that the function of Shg is not to directly anchor the anterior centrosome, but rather to reinforce the polarized organization of the anterior cortex of the pIIa cell. In this view, the function of Shg is similar to its role in polarity establishment in cultured cells. Establishment of adherens junctions in nonpolarized MDCK cells initiates the formation of distinct apical and basal-lateral plasma membrane domains by orienting the delivery of vesicles to a specific cortical site. It is hypothesized that Drosophila Shg may play a similar role in the pIIa cell. Accordingly, Shg would promote the asymmetric targeting of transport vesicles to the anterior lateral membrane and thereby determine the cortical positions of molecules attracting the anterior centrosome (Le Borgne, 2002).

What kind of positional information might be involved in the localized accumulation of Shg-containing complexes in the pIIa cell? One hypothesis is that specific morphological changes in one of the two daughter cells could provide positional information for the division of the other. Accordingly, the formation of the stalk in the pIIb cell would reorganize the anterior cortex of the pIIa cell and determine the position of the cortical patch of Shg in the dividing pIIa cell. However, analysis of the orientation of the pIIa division after inhibition of the formation of the pIIb stalk does not support this view. Alternatively, the point of cytokinesis might provide such positional information. In the budding yeast Saccharomyces cerevisiae, haploid cells divide in a pattern called axial in which the site of division is immediately adjacent to the previous site. A small number of proteins, including the transmembrane protein BUD10, are brought to the site of the previous division. These proteins act as cortical marks required for the axial budding pattern. Recent evidence suggests that BUD10 directly recruits to the bud site the GDP-GTP exchange factor BUD5, which is essential for polarizing the cytoskeleton. This study shows that the pIIa cell divides with the same orientation as the one seen for its mother cell, even when the division axis of the pI cell is randomized, as in a fz mutant background. By analogy to the yeast axial pattern, a cortical mark localizing to the site of cytokinesis from the pI division may be used to orient the pIIa division. This mark would serve to localize Shg and Arm asymmetrically into a small cortical domain in the dividing pIIa cell. This hypothesis remains to be tested (Le Borgne, 2002).

The polar formation of junctional complexes close to the cytokinesis site could constitute a general mechanism to regulate the orientation of an asymmetric cell division relative to the axis of the previous division. For instance, in the Drosophila larval brain, each neuroblast divides asymmetrically in a stem-cell mode with a fixed orientation to generate a series of ganglion mother cells (GMCs), leading to the accumulation of GMCs on one side of the neuroblast. Arm and dAPC2, a Drosophila homolog of the Adenomatous Poliposis Coli protein, colocalize at the cell contact region between the neuroblast and its progeny GMCs. This study raises the possibility that, following the first round of neuroblast division, junctional complexes localizing specifically at the cell-cell contact between the neuroblast and its sister cell may orient the next neuroblast division (Le Borgne, 2002).

The small GTPase Rho is a molecular switch that is best known for its role in regulating the actomyosin cytoskeleton. Its role in the developing Drosophila embryonic epidermis during the process of dorsal closure has been investigated. By expressing the dominant negative DRhoA^N19 construct in stripes of epidermal cells, it has been confirmed that Rho function is required for dorsal closure and it is necessary to maintain the integrity of the ventral epidermis. Defects in actin organization, nonmuscle myosin II localization, the regulation of gene transcription, DE-cadherin-based cell-cell adhesion and cell polarity underlie the effects of DRhoA^N19 expression. Furthermore, these changes in cell physiology have a differential effect on the epidermis that is dependent upon position in the dorsoventral axis. In the ventral epidermis, cells either lose their adhesiveness and fall out of the epidermis or undergo apoptosis. At the leading edge, cells show altered adhesive properties such that they form ectopic contacts with other DRhoA^N19-expressing cells (Bloor, 2002).

Co-expression of RhoA^N19 and GMA, an actin marker in which GFP is fused to the Drosophila Moesin actin-binding domain, demonstrates that, in addition to effects on the actin cytoskeleton, inhibition of RhoA has profound effects on the adhesive properties of epidermal cells. In accordance with this, DE-cadherin is lost from the surface of RhoA^N19-expressing cells. Rho is required for E-cadherin-mediated epithelial cell-cell adhesion in cultured vertebrate cells: in keratinocytes and MDCK cells, blocking Rho function prevents formation of E-cadherin-based junctions and causes preformed junctions to breakdown. This effect is dependent on cell-cell junction maturity; blocking Rho causes E-cadherin to be lost rapidly (within 1 hour) from immature junctions, but E-cadherin can persist for several hours at mature junctions. This differential affect is also observed in this study, since removal of DE-cadherin from the cell surface is not uniform throughout the RhoA^N19-expressing stripe; ventral cells lose surface staining sooner than dorsal cells. This phenomenon most probably reflects regional differences in the maturity of epidermal cell junctions. Cells of the dorsal epidermis form a compact epithelium early in stage 10, while neuroblast delamination in the ventral neurectoderm delays formation of the ventral epidermis proper until well into stage 11, by which time enGAL4- and prdGAL4-driven protein expression is apparent. Alternatively, the differential effect might be due to a dorsoventral gradient in enGAL4- or prdGAL4-driven expression of RhoA^N19. This seems unlikely, since no regional differences in fluorescence are observed when these GAL4 lines are used to drive GMA expression (Bloor, 2002).

If the primary defect associated with the epidermal expression of RhoA^N19 is loss of DE-cadherin, then the phenotypes induced by RhoA^N19 should phenocopy those of shotgun (DE-cadherin) mutants. The defects exhibited by shg embryos are difficult to compare with those shown by embryos expressing RhoA^N19 in epidermal stripes. However, genetic analysis of shg demonstrates that the embryonic dorsal epidermis is less sensitive than the presumptive ventral epidermis to a reduction in DE-cadherin levels; embryos mutant for null shg alleles are missing head and ventral cuticle, while the dorsal cuticle appears unaffected. Thus, as with epidermal expression of RhoA^N19, shg mutants disrupt ventral epidermal integrity and cells undergo apoptosis, while dorsally epidermal cells remain adherent and secrete cuticle. However, there is at least one clear distinction between the genetic reduction of DE-cadherin and the defects induced by expression of RhoA^N19: both null and dominant-negative mutations in shg do not affect epithelial cell polarity, while inhibition of RhoA activity does. It seems likely that this difference reflects the additional function of RhoA in generating cell polarity, possibly through organization of the actin cytoskeleton. It is concluded that the epidermal defects caused by RhoA^N19 expression cannot be explained simply on the basis of loss of DE-cadherin mediated adhesion (Bloor, 2002).

Maintenance of dorsal epithelial integrity in the absence of detectable surface DE-cadherin suggests that a secondary adhesion system must function in the dorsal epidermis. As in vertebrate cells different classical cadherins exhibit cell type dependent sensitivity to Rho inhibition this could involve another member of the cadherin family. The possibility that two cell-cell adhesion systems function in the dorsal epidermis may explain the behavior of RhoA^N19-expressing cells. Embryos that express RhoA^N19 in epidermal stripes differ from shg mutants in the uniformity of DE-cadherin loss from the cell surface. In shg mutants, zygotic DE-cadherin is lost from all cells, while striped expression of RhoA^N19 results in two populations of dorsal epidermal cells -- those with cell surface DE-cadherin and those without. Differential adhesion properties of cell populations are the molecular basis for the classical phenomenon of cell sorting. Thus, the ectopic cell bridges formed by RhoA^N19-expressing cells in these experimental embryos could be due to activation of a cell sorting mechanism between populations of dorsal epidermal cells that associate via different adhesion molecules (Bloor, 2002).

The embryonic cuticle of Drosophila is deposited by the epidermal epithelium during stage 16 of development. This tough, waterproof layer is essential for maintaining the structural integrity of the larval body. Mutations in a set of genes required for proper deposition and/or morphogenesis of the cuticle have been characterized. Zygotic disruption of any one of these genes results in embryonic lethality. Mutant embryos are hyperactive within the eggshell, resulting in a high proportion being reversed within the eggshell (the 'retroactive' phenotype), and all show poor cuticle integrity when embryos are mechanically devitellinized. This last property results in embryonic cuticle preparations that appear grossly inflated compared to wild-type cuticles (the 'blimp' phenotype). One of these genes, krotzkopf verkehrt (kkv), encodes the Drosophila chitin synthase enzyme and a closely linked gene, knickkopf (knk), encodes a novel protein that shows genetic interaction with the Drosophila E-cadherin shotgun. Two other known mutants, grainy head (grh) and retroactive (rtv), show the blimp phenotype when devitellinized, and a new mutation, zeppelin (zep), is described that shows the blimp phenotype but does not produce defects in the head cuticle as do the other mutations (Ostrowski, 2002).

shg is provided both maternally and zygotically and is required for oogenesis as well as for subsequent embryonic development. Zygotic loss of shg function produces defects only in those tissues that undergo dramatic morphogenesis, indicating that maternal shg product provides sufficient cadherin-mediated adhesion for most embryonic epithelia but not for those subject to mechanical stress. The embryonic ventral epidermis undergoes substantial cell rearrangements and degenerates in shg mutant embryos, resulting in a fragmented cuticle. shg loss-of-function mutations are completely recessive, but in embryos homozygous for either knk or zep, heterozygosity for shg leads to a fragmented cuticle phenotype similar to that of a shg homozygote. This effect is not observed for kkv, rtv, or grh, the other blimp class genes. Thus knk and zep seem likely to encode proteins that are more broadly involved in epidermal development. In the absence of these gene functions, a single gene dose of shg becomes inadequate for epithelial cell adhesion in the ventral epidermis. The epidermal tissue of the blimp class mutants appears structurally indistinguishable from that of wild-type embryos. Epidermal cell membrane-associated proteins such as Coracle, and dAPC2 show normal localization and reveal no discernible difference between mutant and wild-type embryos in cell size, shape, or number. Thus it remains to be determined what aspect of epidermal cell function is disrupted in knk and zep mutant embryos (Ostrowski, 2002).

Shotgun role in eye morphogenesis

Ommatidial rotation in the Drosophila eye provides a striking example of the precision with which tissue patterning can be achieved. Ommatidia in the adult eye are aligned at right angles to the equator, with dorsal and ventral ommatidia pointing in opposite directions. This pattern is established during disc development, when clusters rotate through 90°, a process dependent on planar cell polarity and rotation-specific factors such as Nemo and Scabrous. Epidermal growth factor receptor (Egfr) signalling is required for rotation, further adding to the manifold actions of this pathway in eye development. Egfr is distinct from other rotation factors in that the initial process is unaffected, but orientation in the adult is greatly disrupted when signalling is abnormal. It is proposed that Egfr signalling acts in the third instar imaginal disc to 'lock' ommatidia in their final position, and that in its absence, ommatidial orientation becomes disrupted during the remodelling of the larval disc into an adult eye. This lock may be achieved by a change in the adhesive properties of the cells: cadherin-based adhesion is important for ommatidia to remain in their appropriate positions. In addition, there is an error-correction mechanism operating during pupal stages to reposition inappropriately oriented ommatidia. These results suggest that initial patterning events are not sufficient to achieve the precise architecture of the fly eye, and highlight a novel requirement for error-correction, and for an Egfr-dependent protection function to prevent morphological disruption during tissue remodelling (Brown, 2003).

Egfr signalling is required for the maintenance through eye development of the correct orientation of ommatidia. It was speculated that rotation may rely at least partly on the adhesive properties of the cells. In an initial attempt to examine this hypothesis, genetic interactions between components of the Egfr pathway and various adhesion molecules were sought. A Star heterozygote, in which Egfr signalling is slightly reduced, was used as a background in which to look for interactions, because this phenotype is very weak, allowing any enhancement of rotational defects to be easily recognized. Halving the dose of alpha-laminin (wing blister) and the integrin ß subunit (myospheroid) does not modify the Star/+ phenotype. In contrast, alleles of E-cadherin (shotgun) shows a significant interaction with Star, with many more misrotated ommatidia. Under the strongest condition, there is also an enhancement of the rare misrecruitment defects seen in Star/+ eyes, but the enhancement of the rotational defect is independent of this by two criteria. First, the rotational defects were only measured in correctly specified ommatidia; and second, the weaker alleles of shotgun affected rotation without enhancing recruitment. On the basis of these results, it is concluded that the control of rotation by Egfr signalling is linked to cadherin-based adhesion (Brown, 2003).

A model that might account for these results is proposed that suggests that the role of Egfr signalling is to establish a 'locking' mechanism that ensures that ommatidia remain in their final orientation. Such a mechanism might be necessary to protect the ommatidia against positional disruption during later events in eye development. Signalling would therefore be required during or at the end of normal rotation in order to set in place this hypothetical 'lock', although defects might not arise until significantly later than this, when processes occur that would cause ommatidia to reorient in the absence of such a lock (Brown, 2003).

Egfr signaling is evolutionarily conserved and controls a variety of different cellular processes. In Drosophila these include proliferation, patterning, cell-fate determination, migration and survival. Evidence is provided for a new role of Egfr signaling in controlling ommatidial rotation during planar cell polarity (PCP) establishment in the Drosophila eye. Although the signaling pathways involved in PCP establishment and photoreceptor cell-type specification are beginning to be unraveled, very little is known about the associated 90° rotation process. One of the few rotation-specific mutations known is roulette (rlt) in which ommatidia rotate to a random degree, often more than 90°. rlt is shown to be a rotation-specific allele of the inhibitory Egfr ligand Argos; modulation of Egfr activity shows defects in ommatidial rotation. The data indicate that, beside the Raf/MAPK cascade, the Ras effector Canoe/AF6 acts downstream of Egfr/Ras and provides a link from Egfr to cytoskeletal elements in this developmentally regulated cell motility process. Evidence is provided for an involvement of cadherins and non-muscle myosin II as downstream components controlling rotation. In particular, the involvement of the cadherin Flamingo, a PCP gene, downstream of Egfr signaling provides the first link between PCP establishment and the Egfr pathway (Gaengel, 2003).

To specifically test the involvement of cytoskeletal elements and adhesion as well as junctional components, candidate genes were tested for dominant interaction of the mild Star rotation phenotype. These genetic data argue for an involvement of E-Cadherin/shotgun, the atypical cadherin Flamingo (Fmi), the adherens junction protein canoe, non-muscle myosin II (zipper), the septin peanut, and capulet, a protein with actin and adenylate cyclase-binding ability (Gaengel, 2003).

Next, the expression of Fmi and Shotgun in ommatidial preclusters was examined during rotation. Strong LOF alleles of Egfr and its signaling components also affect cell proliferation, fate specification and survival, making the analysis of cell adhesion and junctional components in the context of rotation rather difficult. Thus localization of the cadherins and Arm/ß-catenin was examined in imaginal discs of the rotation-specific aos^rlt allele (Gaengel, 2003).

Although the overall expression and localization of Shotgun and Arm/ß-catenin are largely unaffected, the localization of Fmi is changed in aos^rlt discs. In WT, Fmi is initially present apically in all cells of the morphogenetic furrow and subsequently becomes asymmetrically enriched in the R3/R4 precursor pair. In and posterior to column 6, Fmi is expressed at the membrane of R4, and largely depleted from R3 membranes that do not touch R4, forming a horseshoe-like R4-specific pattern. In contrast, in aos^rlt discs, Fmi restriction to the R4 precursor is generally delayed, and often not established even in columns 8-12, where high levels of Fmi are still seen around the apical membrane cortex of R3 and R4. Since Fmi is thought to act as a homophyllic cell-adhesion molecule, its increased presence on R3 membranes should have a direct effect on Fmi localization in neighboring cells and thus possibly the adhesive properties of the precluster. It is worth noting that although Fmi is required during PCP establishment and R3/R4 cell-fate specification, the delay in Fmi restriction to R4 has no significant effect on the R3/R4 cell-fate decision. Although Fmi interacts with Fz and Notch in this context, the R4-specific mDelta-lacZ marker does not differ significantly from WT and adult aos^rlt eyes also display no defects in R3/R4 specification. Thus, it appears that the delay in Fmi localization specifically affects ommatidial rotation, probably through adhesion, and possibly explains the broad range of rotation angles in aos^rlt and other Egfr pathway mutants (Gaengel, 2003).

Thus Egfr/Ras signaling plays a general role in the regulation of ommatidial rotation. Canoe has been identified as an effector of Ras in this context. Although much is known about how ommatidial chirality and the associated R3/R4 cell-fate decision are regulated (Fz/PCP-Notch signaling), no clear link between the mechanistic aspects of ommatidial rotation and Fz/PCP signaling previously existed. This is the first link to be demonstrated between Egfr signaling and PCP genes, namely Fmi. A further connection between Egfr signaling and PCP establishment is provided by Zipper, which acts downstream of Fz/Dsh and Rok in wing PCP and modifies the Star rotation phenotype. The identification of the Egfr pathway and its regulation of Fmi/cadherin-mediated cell adhesion will serve as an important entry point to further such studies (Gaengel, 2003).

Shotgun role in oogenesis

Shotgun (Shg) is an epithelial cadherin in Drosophila, and forms adherens junctions by associating with Armadillo (beta-catenin). To investigate its role in oogenesis, germ-line clones homozygous for a null mutation in shotgut were generated, and their phenotypes examined and compared with those of armadillo (arm) mutants. In the wild-type ovaries, Shotgun is expressed by both the germ-line and somatic derivatives, colocalizing with Armadillo. In shg mutant ovaries, when the mutation is restricted to the germ line, germ cells are rounded, and generate gaps between themselves, suggesting that their surface adhesiveness is either reduced or lost. However, the positioning of germ cells in the egg chamber is normal. Two groups of somatic follicle cells -- the border cells and centripetal follicle cells -- frequently migrate along incorrect pathways, indicating that DE-cadherin is required for their appropriate migration. Notably, the shg phenotypes are distinct from those of arm null mutants. Intercellular adhesion appears to be less severely affected by arm than by the shg mutation, and the actin-based cytoskeleton and cell arrangement are disorganized only in the arm mutants. These findings suggest that Shotgun is critical for cell-cell adhesion, and functional to a certain extent without Armadillo, whereas Armadillo is required for cytoskeletal organization and for the control of cell positioning. It is therefore proposed that the molecular complex of Shotgun and Armadillo that is present in normal cells is endowed with multiple functions derived from each molecule (Oda, 1997).

The anterior-posterior axis of Drosophila oocyte originates early in oogenesis, when one of two pro-oocytes becomes selected to become the oocyte. This process occurs well before Gurken signaling as one of the earliest steps after the formation of the 16 cell cyst originating from a single cystoblast. The anterior-posterior axis originates from two symmetry-breaking steps during early oogenesis. First, one of the two pro-oocytes within the cyst of 16 germline cells is selected to become the oocyte. This cell then comes to lie posterior to the other germline cells of the cyst, thereby defining the polarity of the axis. The oocyte reaches the posterior of the cyst in two steps: (1) the cyst flattens as it enters region 2b of the germarium to place the two pro-oocytes in the center of the cyst, where they contact the posterior follicle cells; (2) one cell is then selected to become the oocyte and protrudes into the posterior follicle cell layer when the cyst rounds up on entering region 3. During this germ cell rearrangement, the components of the homophilic cadherin adhesion complex, DE-cadherin, Armadillo and alpha-catenin, accumulate along the border between the oocyte and the posterior follicle cells. The positioning of the oocyte requires cadherin-dependent adhesion between these two cell types, since the oocyte is frequently misplaced when DE-cadherin, known as Shotgun, is removed from either the germline or the posterior follicle cells. It is concluded that the oocyte reaches the posterior of the germline cyst because it adheres more strongly to the posterior follicle cells than its neighbours during the germ cell rearrangement that occurs as the cyst moves into region 3. The Drosophila anterior-posterior axis therefore becomes polarized by an unusual cadherin-mediated adhesion between a germ cell and mesodermal follicle cells (Gonzalez-Reyes, 1998).

In region 2a, the cysts already contain 16 germ cells and form clusters that are several cells thick and extend only part way across of the germarium. At this stage, Bic-D protein is usually concentrated in the two adjacent pro-oocytes, which occupy random positions within the cyst. These two cells each have 4 ring canals and are connected to one another by the oldest ring canal, which can be distinguished from the others because it stains more strongly with Rhodamine-Phalloidin. When the cyst enters region 2b, it spreads to form a 1-cell-thick disc that extends across the width of the germarium. Bic-D protein has now accumulated in only one of the pro-oocytes, the presumptive oocyte, but the two cells with 4 ring canals occupy equivalent positions in the middle of the cyst. This suggests that the single cell that is fated to become the oocyte has already been determined and has diverged biochemically, from its sister cell, fated to become a nurse cell. At the same time, the somatic follicle cells start to migrate to surround the cyst, and accumulate at its posterior to separate it from the preceding cyst. As the cyst moves down the germarium from region 2b to region 3, the germ cells rearrange to change the shape of the cyst from a flattened disc to a sphere. At the end of this reorganization, the oocyte always lies at the posterior of the cyst and protrudes from the sphere of germ cells into the surrounding follicle cell layer. Thus, the oocyte reaches the posterior during the transition from region 2b to region 3, in a process that seems to involve an interaction between the oocyte and the adjacent follicle cells. The initial organization of the germ cells in region 2b seems to bias the cell rearrangement that occurs during the transition to region 3, so that the two pro-oocytes are more likely to end up at the posterior of the cyst than the other 14 cells. Despite the correct determination of the oocyte, this cell is not correctly positioned in 32% of the cysts. These observations indicate that the oocyte needs to be specified by region 2b to guarantee that it is correctly positioned. This suggests that the oocyte plays an active role in its positioning during the germ cell rearrangement that occurs as the cyst enters region 3, and that this is essential to ensure that the oocyte rather than the losing pro-oocyte comes to lie at the posterior of the cyst (Gonzalez-Reyes, 1998).

The phenotypes produced by armadillo and shotgun mutant germline clones have suggested that Shotgun-mediated adhesion may be required either to localize the oocyte to the posterior of the cyst or to maintain it in this position as the egg chamber grows. To distinguish between these possibilities, germline clones that were mutant for four shotgun alleles of increasing strength were generated. All four mutants give rise to egg chambers with misplaced oocytes, and the penetrance of this phenotype correlates with the severity of the mutant allele. This phenotype is already apparent in the germarium. For example, although the region 2b cysts usually have a wild-type arrangement of germ cells in shg IH germline clones, the oocyte never protrudes into the follicle cell layer when the cyst enters region 3, and frequently occupies a lateral position. An identical phenotype is also seen in germline clones of a strong armadillo allele. Thus, Shotgun and Armadillo are required in the germ cells for the initial positioning of the oocyte at the posterior of the cyst during the cell rearrangement that takes place as the cyst moves into region 3. In addition to producing a very high frequency of misplaced oocytes in region 3, germline clones of shg IG29 disrupt the organization of the germ cells earlier in the germarium. The mutant cysts do not flatten in region 2b to form the 1-cell-thick disc that extends across the width of the germarium, and remain the same shape as region 2a cysts. shotgun mutants do not affect oocyte determination. Most known mutants that affect oocyte positioning seem to do so indirectly by disrupting the determination and differentiation of the oocyte. However, this does not appear to be case for shotgun and armadillo mutants, since all of the markers for oocyte differentiation examined are expressed normally in the misplaced oocytes produced by germline clones (Gonzalez-Reyes, 1998).

Although Shotgun becomes enriched in the most anterior follicle cells, the highest levels are seen along the boundary between the oocyte and the posterior follicle cells as the cyst moves from region 2b to region 3. This accumulation disappears once the cyst has left the germarium, however, although the two polar follicle cells continue to express higher levels of Shotgun throughout oogenesis. Armadillo and alpha-catenin show an identical transient concentration at the junction between the oocyte and the posterior follicle cells. The co-localization of three components of the cadherin adhesion complex to this boundary strongly supports a model in which the localization of the oocyte is driven by an increase in cadherin-dependent adhesion between the oocyte and these specific somatic cells. To determine the relative contributions of the oocyte and the follicle cells to this posterior enrichment, the distribution of Shotgun was examined in egg chambers that contain shotgun mutant germline clones. Despite the lack of Shotgun in the germline and the mispositioning of the oocyte, the protein is still concentrated at the posterior of these cysts in the follicle cell membranes that face the germ cells. Shotgun therefore accumulates in these apical membranes, even when these cells do not contact the oocyte and there is no Shotgun in the adjacent germ cells. The positioning of the oocyte is shown to be disrupted when the posterior follicle cells lack shotgun (Gonzalez-Reyes, 1998).

The cadherin family of adhesion molecules generally mediate homophilic adhesion between cells of the same type, and this is also the case for the Shotgun-dependent adhesion between the germ cells during the flattening of the cyst in region 2b, the first of two steps in which Shotgun is involved in the correct placement of the oocyte. The differential adhesion between the oocyte and the posterior follicle cells that occurs at the next stage is quite different, however, because these two cell types are completely unrelated and arise from separate lineages that are set aside at the earliest stages of embryogenesis. The oocyte is descended from the pole cells, which are the primordial germ cells that form at the posterior of the embryo about one and a half hours after fertilization, whereas the follicle cells arise from the gonadal mesoderm. This role for cadherin in heterotypic adhesion is very unusual, but not entirely without precedent. In mammals, E-cadherin has been shown to mediate adhesion between Langerhans cells and keratinocytes, while N-cadherin contributes to the attachment between developing spermatocytes and the Sertoli cells of the testis. It is interesting to note that the latter example also involves adhesion between germline and somatic cells (Gonzalez-Reyes, 1998 and references).

In most animal species, germ cells require intimate contact with specialized somatic cells in the gonad for their proper development. The establishment of germ cell-soma interaction during embryonic gonad formation has been analyzed in Drosophila; somatic cells undergo dramatic changes in cell shape and individually ensheath germ cells as the gonad coalesces. Germ cell ensheathment is independent of other aspects of gonad formation, indicating that separate morphogenic processes are at work during gonadogenesis. The cell-cell adhesion molecule Drosophila E-cadherin is essential both for germ cell ensheathment and gonad compaction, and is upregulated in the somatic gonad at the time of gonad formation. Differential cell adhesion contributes to cell sorting and the formation of proper gonad architecture. In addition, Fear of Intimacy, a novel transmembrane protein, is also required for both germ cell ensheathment and gonad compaction. E-cadherin expression in the gonad is dramatically decreased in fear of intimacy mutants, indicating that Fear of Intimacy may be a regulator of E-cadherin expression or function (Jenkins, 2003).

Gonad coalescence in Drosophila is the rearrangement of germ cells and somatic gonadal precursors (SGPs) from a broad association stretching across three parasegments (10, 11 and 12) of the embryo to a tight cluster of cells located in PS10. In this process, germ cells become enclosed in the environment that will nurture them as they adopt stem cell fates and begin gametogenesis. Germ cell-soma contact in the embryonic gonad is already extensive, with each germ cell becoming surrounded by somatic cell membrane. Furthermore, E-cadherin plays a key role in this and other aspects of gonad formation. Detailed analysis of gonad coalescence has shown that it can be subdivided into two processes: gonad compaction and germ cell ensheathment. In gonad compaction, SGPs and germ cells physically condense together to create a rounded organ. Germ cell ensheathment is characterized by the dramatic shape changes of SGPs that produce thin cellular extensions that surround the germ cells. Germ cells lack cellular extensions during gonad compaction, and need not be present for compaction to occur. This suggests that SGPs provide the 'driving force' behind the movements of compaction and germ cells play a more passive role (Jenkins, 2003).

Several pieces of data indicate that gonad compaction and germ cell ensheathment are distinct, separable events. Germ cell ensheathment is already apparent at stage 13, prior to the onset of compaction. In addition, compaction proceeds normally in agametic embryos, despite a lack of germ cell ensheathment. Furthermore, in mutants that affect gonad coalescence (shg, foi), examples of gonads with no ensheathment but a high degree of compaction have been observed, and also gonads with good ensheathment but little compaction. Thus, gonad compaction and germ cell ensheathment are independent processes that together contribute to the proper architecture of the coalesced embryonic gonad. Both of these processes require the adhesion molecule E-cadherin (Jenkins, 2003).

How might Drosophila E-cadherin function to promote gonad morphogenesis? Differential cell adhesion mediated by E-cadherin has been shown to govern cell sorting in vitro and in at least one in vivo situation. It is possible to explain these observations of gonad morphogenesis with a similar model of differential cell adhesion. In this model, gonad compaction results from an increased affinity of SGPs for one another relative to the surrounding mesoderm. Compaction would occur as SGPs maximize their contacts with one another and minimize their contacts with the surrounding mesoderm, hence forming a sphere. Contacts between SGPs and germ cells might also play a role in compaction, but SGP-SGP affinity would be sufficient to allow this process to occur in the absence of germ cells. Consistent with this hypothesis, E-cadherin expression becomes more apparent in SGPs relative to the surrounding mesoderm at the time that compaction is initiated. This is likely to reflect an increase in E-cadherin expression or stability, but could also conceivably result from a change in subcellular localization. Upregulation of E-cadherin in the SGPs may contribute to an increase in SGP-SGP adhesion during gonad compaction (Jenkins, 2003).

The process of germ cell ensheathment may also be controlled by differential cell adhesion, but between SGPs and germ cells. Ensheathment would occur as a result of SGPs maximizing their contacts with germ cells. This model requires that SGPs and germ cells have a higher affinity for each other than for their own cell type. A prediction of this model is that ensheathment would be blocked if germ cell-germ cell adhesion were increased, which is exactly what is observed (Jenkins, 2003).

What role might E-cadherin, traditionally a homophilic cell adhesion molecule, play in mediating the heterotypic interactions between SGPs and germ cells during ensheathment? One possibility is the presence of additional heterophilic adhesion molecules that promote specific adhesion between these cell types. A candidate member of such a heterophilic adhesion system is Neurotactin, which is present on SGPs and has been shown to promote heterotypic cell adhesion. In this case, E-cadherin could provide additional 'glue' that is required for ensheathment once the heterotypic specificity between SGPs and germ cells is established. Alternatively, E-cadherin might somehow be biased to act in a heterophilic manner. E-cadherin could interact with a heterophilic binding partner, or could be biased by a modification or co-factor to bind preferentially to E-cadherin molecules on heterotypic cells (e.g. a modified form of E-cadherin might interact only with an unmodified form) (Jenkins, 2003).

Gonad coalescence may represent an elegant example of organogenesis based on differential cell adhesion. A hierarchy of cell affinity (SGP-germ cell>SGP-SGP>SGP-surrounding mesoderm) can account for much of the observed gonad organization. This model requires cell movement for proper execution of cell sorting. Although the morphology of the germ cells suggests that they may not be highly motile at this time, further work is needed to determine the extent to which SGP versus germ cell movement contributes to this process. In addition, other mechanisms, such as cytoskeletal-derived contractile and protrusive forces, may also be important for compaction and ensheathment. The contribution of these different factors to the overall architecture of the gonad can now be further tested using a more detailed understanding of gonad coalescence (Jenkins, 2003).

Embryos with mutations in the fear of intimacy gene share several gonad defects with shg mutant embryos, including defects in gonad compaction and germ cell ensheathment. Both genes are also required for tracheal branch fusion, suggesting that Drosophila E-cadherin and FOI may work together to promote all of these processes. Consistent with this, E-cadherin protein levels are severely reduced within the gonads of foi mutants. E-cadherin expression is reduced in SGPs, which display defective behaviors in foi mutants. Thus, gonad defects in foi mutants correlate strongly with the cells in which E-cadherin expression is most affected, suggesting that this may be the cause of the foi mutant phenotype (Jenkins, 2003).

There are several possible models for how FOI, a cell surface, multipass transmembrane protein, might be affecting the levels of E-cadherin protein. FOI could act as a receptor or channel that signals the beginning of coalescence. Upregulation of E-cadherin in the SGPs could require such a signal. Or, FOI might act to localize E-cadherin complexes to sites of germ cell-soma and soma-soma contact within the gonad. As such, FOI could act during the export of E-cadherin to the cell surface, or to localize E-cadherin to specific sites of cell-cell contact. Alternatively, FOI might affect E-cadherin levels by affecting its function as a cell adhesion molecule. It has been suggested that the stability of E-cadherin is tightly linked to its function in adhesion complexes, with reduced E-cadherin function leading to a faster turnover of the protein. FOI might modulate E-cadherin function by acting as a co-factor itself on the cell surface, or by acting as a transporter to alter the concentration of a small molecule modulator of E-cadherin adhesion, such as Ca²⁺ (Jenkins, 2003).

Germ cell ensheathment in the Drosophila embryonic gonad is an example of a recurring theme in germ cell development; germ cells require close contact with specialized somatic cells for their proper differentiation. Germ cell-soma interaction has been shown to be essential for many phases of germ cell development in diverse species. The proper sexual identity of the germline is controlled by the soma in both the mouse and the fly. In addition, germ cells often exist as stem cells in the adult gonad, dividing to produce one daughter that enters gametogenesis while the other retains stem cell identity. Interaction between germline stem cells and their somatic niche is essential for regulating cell division and stem cell maintenance. Finally, during gametogenesis, differentiating germ cells remain in close association with somatic cells that regulate their development into sperm or egg (Jenkins, 2003).

Adhesive contacts and cell-cell junctions are crucial for soma-germline signaling. Some somatic signals require specific cellular junctions, such as gap junctions. Even secreted signals, such as those governing the regulation of germline stem cell maintanence, require the proper adhesion and orientation between germline and soma. E-cadherin has been shown to play a crucial role in several examples of germ cell-soma interaction, including in the stem cell niche and developing egg chamber in Drosophila (Jenkins, 2003).

Regulation of germ cell development by the soma may begin as soon as the gonad forms. There is evidence that the soma regulates sex determination and the cell cycle in the mouse germline and the pattern of germ cell gene expression in Drosophila at very early stages. Thus, regulation by the soma is crucial for every stage of germ cell development. It is hypothesized that the E-cadherin-dependent germ cell ensheathment observed in embryonic gonads creates a nascent niche that allows the SGPs to regulate germ cell development and the transition to germline stem cells (Jenkins, 2003).

Rho1 regulates Drosophila adherens junctions independently of p120ctn: Genetic interactions with shotgun

During animal development, adherens junctions (AJs) maintain epithelial cell adhesion and coordinate changes in cell shape by linking the actin cytoskeletons of adjacent cells. Identifying AJ regulators and their mechanisms of action are key to understanding the cellular basis of morphogenesis. Previous studies (Magie, 2002) linked both p120catenin and the small GTPase Rho to AJ regulation and revealed that p120 may negatively regulate Rho. This study examined the roles of these candidate AJ regulators during Drosophila development. It was found that although p120 is not essential for development, it contributes to morphogenesis efficiency, clarifying its role as a redundant AJ regulator. Rho has a dynamic localization pattern throughout ovarian and embryonic development. It preferentially accumulates basally or basolaterally in several tissues, but does not preferentially accumulate in AJs. Further, Rho1 localization is not obviously altered by loss of p120 or by reduction of core AJ proteins. Genetic and cell biological tests suggest that p120 is not a major dose-sensitive regulator of Rho1. However, Rho1 itself appears to be a regulator of AJs. Loss of Rho1 results in ectopic accumulation of cytoplasmic DE-cadherin, but ectopic cadherin does not accumulate with its partner Armadillo. These data suggest Rho1 regulates AJs during morphogenesis, but this regulation is p120 independent (Fox, 2005).

Analysis of Rho1 and Rho1 shg mutants is consistent with the hypothesis that Rho1 regulates AJs, but suggests that their interactions are complex. A weak shg allele was enhanced, but stronger alleles were suppressed. There are several possible explanations for these contrasting results. Weak alleles (e.g. shg^G119) make protein with reduced but residual function. If Rho1 negatively regulates cadherin endocytosis, more mutant DE-Cad protein might be endocytosed in Rho1's absence, further reducing functional DE-Cad and enhancing the phenotype. However, null or very strong shg alleles accumulate no functional DE-Cad at AJs, rendering regulation of cadherin endocytosis a moot point. The slight suppression by Rho1 of strong shg alleles may result from a reduction of morphogenetic movements, reducing cuticle disruption. Alternatively, some mutant DE-Cad proteins may be capable of coupling to Rho1 while others are not. Rho1 can bind alpha-catenin (Magie, 2002), and active Rho1 may be recruited to AJs by that interaction. shg^G119 has a wild-type cytoplasmic domain and could presumably couple to Rho1; reducing Rho1 might further impair its function. By contrast, the shg² mutation may impair Arm and/or alpha-catenin binding and thus Rho1 recruitment; if so this mutant protein would not be further impaired by Rho1 removal. Finally, the complex genetic interactions might reflect different requirements for Rho1 during neuroblast delamination and head involution, which are affected by strong or weak reduction in DE-Cad function, respectively. Future studies of Rho regulation of and by AJs will help distinguish between these possibilities (Fox, 2005).

Shotgun and tumorogenesis; Loss of cell polarity drives tumor growth and invasion through JNK activation in Drosophila

Apparent defects in cell polarity are often seen in human cancer. However, the underlying mechanisms of how cell polarity disruption contributes to tumor progression are unknown. Using a Drosophila genetic model for Ras-induced tumor progression, a molecular link has been shown between loss of cell polarity and tumor malignancy. Mutation of different apicobasal polarity genes activates c-Jun N-terminal kinase (JNK) signaling and downregulates the E-cadherin/β-catenin adhesion complex, both of which are necessary and sufficient to cause oncogenic Ras^V12-induced benign tumors in the developing eye to exhibit metastatic behavior. Furthermore, activated JNK and Ras signaling cooperate in promoting tumor growth cell autonomously, since JNK signaling switches its proapoptotic role to a progrowth effect in the presence of oncogenic Ras. The finding that such context-dependent alterations promote both tumor growth and metastatic behavior suggests that metastasis-promoting mutations may be selected for based primarily on their growth-promoting capabilities. Similar oncogenic cooperation mediated through these evolutionarily conserved signaling pathways could contribute to human cancer progression (Igaki, 2006).

Most human cancers originate from epithelial tissues. These epithelial tumors, except for those derived from squamous epithelial cells, normally exhibit pronounced apicobasal polarity. However, these tumors commonly show defects in cell polarity as they progress toward malignancy. Although the integrity of cell polarity is essential for normal development, how cell polarity disruption contributes to the signaling mechanisms essential for tumor progression and metastasis is unknown. To address this, a recently established Drosophila model of Ras-induced tumor progression triggered by loss of cell polarity has been used. This fly tumor model exhibits many aspects of metastatic behaviors observed in human malignant cancers, such as basement membrane degradation, loss of E-cadherin expression, migration, invasion, and metastatic spread to other organ sites (Pagliarini, 2003). In the developing eye tissues of these animals, loss of apicobasal polarity is induced by disruption of evolutionarily conserved cell polarity genes such as scribble (scrib), lethal giant larvae (lgl), or discs large (dlg), three polarity genes that function together in a common genetic pathway, as well as other cell polarity genes such as bazooka, stardust, or cdc42. Oncogenic Ras (Ras^V12), a common alteration in human cancers, causes noninvasive benign overgrowths in these eye tissues (Pagliarini, 2003). Loss of any one of the cell polarity genes somehow strongly cooperates with the effect of Ras^V12 to promote excess tumor growth and metastatic behavior. However, on their own, clones of scrib mutant cells are eliminated during development in a JNK-dependent manner; expression of Ras^V12 in these mutant cells prevents this cell death (Igaki, 2006).

To better quantify the metastatic behavior of tumors in different mutant animals, the analysis focused on invasion of the ventral nerve cord (VNC), a process in which tumor cells leave the eye-antennal discs and optic lobes (the areas where they were born) and migrate to and invade a different organ, the VNC. It was further confirmed that the genotypes associated with the invasion of the VNC in this study also resulted in the presence of secondary tumor foci at distant locations, although the number and size of these foci were highly variable (Pagliarini, 2003; Igaki, 2006).

In analyzing the global expression profiles of noninvasive and invasive tumors induced in Drosophila developing eye discs, it was observed that expression of the JNK phosphatase puckered (puc) was strongly upregulated in the invasive tumors. Upregulation of puc represents activation of the JNK pathway in Drosophila. Therefore an enhancer-trap allele, puc-LacZ, was used to monitor the activation of JNK signaling in invasive tumor cells. Strong ectopic JNK activation was present in invasive tumors, while only a slight expression of puc was seen in restricted regions of Ras^V12-induced noninvasive overgrowth. Intriguingly, more intense JNK activation was seen in tumor cells located in the marginal region of the eye-antennal disc and tumor cells invading the VNC. Analysis of clones of cells with a cell polarity mutation alone revealed that JNK signaling was activated by mutation of cell polarity genes. Notably, JNK signaling was not activated in a strictly cell-autonomous fashion. JNK activation in these cells was further confirmed by anti-phospho-JNK antibody staining that detects activated JNK (Igaki, 2006).

To examine the contribution of JNK activation to metastatic behavior, the JNK pathway was blocked by overexpressing a dominant-negative form of Drosophila JNK (Bsk^DN). As previously reported (Pagliarini, 2003), clones of cells mutant for scrib, lgl, or dlg do not proliferate as well as wild-type clones, while combination of these mutations with Ras^V12 expression resulted in massive and metastatic tumors. Strikingly, inhibition of JNK activation by Bsk^DN completely blocked the invasion of the VNC, as well as secondary tumor foci formation. Drosophila has two homologs of TRAF proteins (DTRAF1 and DTRAF2), which mediate signals from cell surface receptors to the JNK kinase cascade in mammalian systems. It was found that RNAi-mediated inactivation of DTRAF2, but not DTRAF1, in the tumors strongly suppressed their metastatic behavior. Inactivation of dTAK1, a Drosophila JNK kinase kinase (JNKKK), or Hep, a JNKK, also suppressed metastatic behavior. Drosophila has two known cell surface receptors that act as triggers for the JNK pathway, Wengen (TNF receptor) and PVR (PDGF/VEGF receptor). Intriguingly, it was found that RNAi-mediated inactivation of Wengen partially suppressed tumor invasion. Inactivation of PVR, in contrast, did not show any suppressive effect on metastatic behavior. It was also found that the metastatic behavior of Ras^V12-expressing tumors that were also mutated for one of three other cell polarity genes, bazooka, stardust, or cdc42, was also blocked by Bsk^DN. These data indicate that loss of cell polarity contributes to metastatic behavior by activating the evolutionarily conserved JNK pathway (Igaki, 2006).

Next, whether JNK activation is sufficient to trigger metastatic behavior in Ras^V12-induced benign tumors was examined. Two genetic alterations can be used to activate JNK in Drosophila. First, JNK signaling can be activated by overexpression of Eiger, a Drosophila TNF ligand. While mammalian TNF superfamily proteins activate both the JNK and NFκB pathways, Eiger has been shown to specifically activate the JNK pathway through dTAK1 and Hep. Indeed, the eye phenotype caused by Eiger overexpression could be reversed by blocking JNK through Bsk-IR (Bsk-RNAi). Second, overexpression of a constitutively activated form of Hep (Hep^CA) can also activate JNK signaling. However, the eye phenotype caused by Hep^CA overexpression was only slightly suppressed by Bsk-IR, suggesting that Hep^CA overexpression may have additional effects other than JNK activation. Therefore Eiger overexpression was used to activate JNK in Ras^V12-induced benign tumors, and it was found that the Ras^V12+Eiger-expressing tumor cells did not result in the invasion of the VNC. This indicates that loss of cell polarity must induce an additional downstream effect(s) essential for metastatic behavior. A strong candidate for the missing event is downregulation of the E-cadherin/catenin adhesion complex, since this complex is frequently downregulated in malignant human cancer cells and is also downregulated by loss of cell polarity genes in Drosophila invasive tumors (Pagliarini, 2003). In addition, it has been recently reported that higher motility of mammalian scrib knockdown cells can be partially rescued by overexpression of E-cadherin-catenin fusion protein, suggesting a role of E-cadherin in preventing polarity-dependent invasion. Furthermore, overexpression of E-cadherin blocks metastatic behavior of Ras^V12/scrib^−/− tumors (Pagliarini, 2003), indicating that loss of E-cadherin is essential for inducing tumor invasion in this model. It was found that loss of the Drosophila E-cadherin homolog shotgun (shg), combined with the expression of both Ras^V12 and Eiger, induced the invasion of the VNC. Intriguingly, loss of shg in Ras^V12-expressing clones also showed a weak invasive phenotype at lower penetrance. In agreement with the essential role of JNK in tumor invasion, clones of shg^−/− cells weakly upregulated puc expression. It was further found that JNK activation in dlg^−/− clones is not blocked by overexpression of E-cadherin, suggesting that mechanism(s) other than loss of E-cadherin also exist for inducing JNK activation downstream of cell polarity disruption. The metastatic behavior of Ras^V12+Eiger/shg^−/− tumors was completely blocked by coexpression of Bsk^DN, indicating a cell-autonomous requirement of JNK activation for this process. Furthermore, it was found that loss of the β-catenin homolog armadillo also induced metastatic behavior in Ras^V12-induced benign tumors. In contrast, overexpression of Hep^CA in Ras^V12/shg^−/− cells resulted in neither enhanced tumor growth nor metastatic behavior. Together, these results suggest that, although the Ras^V12+Eiger/shg^−/− does not completely phenocopy the effect of Ras^V12/scrib^−/−, activation of JNK signaling and inactivation of the E-cadherin/catenin complex are the downstream components of cell polarity disruption that trigger metastatic behavior in Ras^V12-induced benign tumors (Igaki, 2006).

Dcas supports cell polarization and cell-cell adhesion complexes in development

Mammalian Cas proteins regulate cell migration, division and survival, and are often deregulated in cancer. However, the presence of four paralogous Cas family members in mammals (BCAR1/p130Cas, EFS/Sin1, NEDD9/HEF1/Cas-L, and CASS4/HEPL) has limited their analysis in development. The single Drosophila Cas gene, Dcas, was deleted to probe the developmental function of Dcas. Loss of Dcas had limited effect on embryonal development. However, Dcas is an important modulator of the severity of the developmental phenotypes of mutations affecting integrins (If and mew) and their downstream effectors Fak56D or Src42A. Strikingly, embryonic lethal Fak56D-Dcas double mutant embryos had extensive cell polarity defects, including mislocalization and reduced expression of E-cadherin. Further genetic analysis established that loss of Dcas modified the embryonal lethal phenotypes of embryos with mutations in E-cadherin (Shg) or its signaling partners p120- and beta-catenin (Arm). These results support an important role for Cas proteins in cell-cell adhesion signaling in development (Tikhmyanova, 2010).

This work identifies a strong interaction between the Dcas, and integrin pathway genes, including integrins and their effector kinases Fak56D and Src42A, during early embryonal development in Drosophila. The synthetic lethal phenotypes found in double mutants of Dcas and Src or FAK56D were marked by defects in dorsal closure and in some cases by the appearance of anterior cuticle holes that suggested head involution defects. These defects were commonly accompanied by abnormalities in epithelial function, including failure to appropriately localize shg/E-cadherin to cell junctions, and reduced shg expression. The data are compatible with the idea that either Fak56D or Dcas is sufficient to support shg/E-cadherin localization and cell polarization during morphogenetic movements in Drosophila embryos, but the absence of both cannot be sustained (Tikhmyanova, 2010).

Building from these observations, a novel synthetic lethal relationship was established between DCas, shg, and arm. As with crosses to alleles of Fak56D and Src42A, the point of lethality was at the time of dorsal closure, at embryonal stages 15-16, and associated with defective cuticle formation. One way to integrate these observations is to hypothesize that the DCas, Fak56D, and Shg protein products are normally in dynamic balance, with Dcas regulating Shg cycling. The fact that Crb and Dlg1, a mammalian homolog of Dlg, have been reported to support Shg localization to adherens junctions, suggests that Dcas/Fak56/Src42A specifically interact to support this cell polarity/cell junctional control system. In this context, it is suggestive that the Crb family protein CRB3 has been described as part of a complex including CRB3, Pals1, and PatJ that becomes tightly associated with Src kinase during reorganization of cell polarity. In the absence of DCas and Fak56D, Shg cannot localize properly; the moderately elevated levels of Shg proteins found in these embryos most likely arises as part of a cellular compensatory mechanism in response to decreased functional Shg signaling complexes. In further indirect support of the idea that this is a specific Dcas action, the fact that genetic interactions were not observed between Dcas¹ and Aur or Dock indicates that Dcas does not promiscuously interact with other genetic lesions to reduce viability (Tikhmyanova, 2010).

A previous study demonstrated a role for Dcas in axonal guidance in the development of the nervous system of adult flies (Huang, 2007). That work analyzed the hypomorphic Dcas mutant allele Dcas^P1, and the small deficiency Df(3L)Exel6083, including Dcas and five adjacent genes, which were also used in this study. The earlier study focused exclusively on analyzing the contribution of Dcas to axonal guidance in late (stage 16/17) embryos: in that analysis, Dcas functioned similarly to integrins, and genetically interacted with integrins (if, mew, and mys) in regulating axon guidance and axonemal defasciculation. In this context, it is intriguing that the mammalian Cas family NEDD9 gene is abundant during neuronal development, has been proposed as a candidate locus for oral cleft defects in humans based on its chromosomal location near the OFC-1 locus. Together these findings raise the possibility that this specific Dcas paralog has a specific role in human neuronal migration and morphogenesis of the head. As with the current data using the new Dcas¹ allele, homozygous deletion of Dcas in conjunction with integrins had moderate effect on viability of adult flies, although this work for the first time demonstrates an interaction between Dcas and if and mew, and also between Dcas and Src, in regulation of wing development (Tikhmyanova, 2010).

Generation of the first null allele of Dcas provides a useful new tool to study the role of this protein in Drosophila development. This work illuminates the evolutionary conservation of Dcas function within the integrin and receptor tyrosine kinase network, including FAK, Src, and integrins genes. The finding that a low percentage of embryos with mutant Dcas and all embryos with double mutations in Dcas and Fak56D, have perturbed localization of polarity markers, including Shg, indicates a novel function for Cas family in regulation of cell polarity. To date, the evidence directly connecting Cas proteins to a known mechanism for control of cell polarity is sparse. Although NEDD9 was in fact discovered in a functional genomics screen for cell cycle and polarity modifiers in budding yeast (leading to its designation as HEF1, Human Enhancer of Filamentation 1), the mechanism involved was not established, and given the great evolutionary distance involved, may not be relevant to a role in metazoans. Both BCAR1 and NEDD9 interact physically with proteins that influence cell polarity controls during pseudopod extension and other actin polarization processes: these include the GTP exchange factor AND-34 (Tikhmyanova, 2010).

The data in the present study indicating genetic interactions with cell-cell junction regulatory proteins Shg, Arm and p120-catenin may have considerable significance in the sphere of cancer research, as it implies that overexpression of Cas proteins may promote cancer progression by influencing the polarized movement of cells and influencing lateral attachments. The fact that one report has indicated interactions between BCAR1 and nephrocystins at cell-cell junctions in polarized epithelial cells implies that a potentially direct interaction of Cas proteins in these structures is conserved through mammals. However, given the known interactions of Cas proteins with FAK and SRC at focal adhesions, another possibility is that Cas may additionally or alternatively impact Shg function through indirect signaling emanating from these structures. Notably, it has been reported that NEDD9 overexpression induced by dioxin caused downregulation of E-cadherin, and it will be of great interest to study the consequences of overexpressing Dcas on Drosophila development. Consequences for loss of NEDD9 expression on E-cadherin expression or localization are not yet known. Resolving these questions will provide intriguing directions for future studies (Tikhmyanova, 2010).

Cell migration in Drosophila optic lobe neurons is controlled by eyeless/Pax6

In the developing Drosophila optic lobe, eyeless, apterous and distal-less, three genes that encode transcription factors with important functions during development, are expressed in broad subsets of medulla neurons. Medulla cortex cells follow two patterns of cell movements to acquire their final position: first, neurons are arranged in columns below each neuroblast. Then, during pupation, they migrate laterally, intermingling with each other to reach their retinotopic position in the adult optic lobe. eyeless, which encodes a Pax6 transcription factor, is expressed early in progenitors and controls aspects of this cell migration. Its loss in medulla neurons leads to overgrowth and a failure of lateral migration during pupation. These defects in cell migration among medulla cortex cells can be rescued by removing DE-Cadherin. Thus, eyeless links neurogenesis and neuronal migration (Morante, 2011).

Medulla cortex cells indeed originate from the OPC. In adults, there are ~800 'columns' in the medulla, each formed by more than 60 different cell types that repetitively contact every R7-R8 fascicle (Morante, 2008). The spatial segregation of ey-, ap- and dll-positive cells observed early in larval brain lobes is lost during pupal development through two modes of cell movements happening during larval (radial) and pupal life (mostly lateral). As a result, each adult 'column' in wild-type medulla is composed of the precise complement of neurons necessary to process visual information. Each R7/R8 termination pair is surrounded by at least 47 different cell types, while 13 other cell types do not appear to contact PRs (Morante, 2008). Further experiments will determine whether the cell movements shown by medulla cortex cells during optic lobe organogenesis involve the dispersion of the entire neuron, or only the cell body (Morante, 2011).

ey-, dll- and ap-positive neurons originating from the OPC-derived neuroblasts show a radial organization, while the number of medulla neurons expands. This organization resembles the radial units present in the embryonic mammalian neocortex where cells reach their appropriate layer via radial migration along glial processes, expanding the size of the cerebral cortex . However, it is unlikely that there is active radial migration in the medulla, as neurons appear to be simply displaced by newly formed neurons above them (Morante, 2011).

The lack of eyeless causes an increase in neuroblast proliferation and impedes cells from reaching their final position in the medulla. It should be noted that some ey^DN cells still manage to disperse in the medulla cortex. They might represent cells that express ey-Gal4 but do not express or require ey for their migration, or cells that express lower levels of ey-Gal4 in which ey^DN is not sufficient to affect function (Morante, 2011).

The effect of ey^DN was examined on eye imaginal discs, and the same phenotype was observed as that reported in a previous study; expression of ey^DN resulted in underproliferation of eye imaginal disc cells and a partial or complete loss of eye structure, which is similar to what is seen in eyeless mutants. Thus, the effects of ey^DN in the eye disc are, surprisingly, the opposite of what was observed in medulla neuroblasts (Morante, 2011).

Furthermore, to address a possible role of eyeless in postmitotic cells, ey^DN was mis-expressed using an act-FRT-stop-FRT-Gal4 construct both in larvae and in postmitotic cells (after P25). This mis-expression resulted in lethality, even with very short heat-shocks, probably owing to the strength of the act-Gal4 driver; this precludes analysis in adults. Thus, at this point, a possible role of eyeless in postmitotic cells cannot be eliminated (Morante, 2011).

Interestingly, Sey mice embryos, which are mutant for the mouse ortholog of ey (Pax6), exhibit an increase in the number of Cajal-Retzius cells, which migrate tangentially. Simultaneously, late-born cortical precursors show abnormalities in their patterns of radial migration, forming heterotopic clusters in the path towards their final position in the embryonic cerebral cortex. Although this defective neuronal radial migration has been proposed to be due to an altered radial glia morphology, other studies have shown that these clusters exhibit an increase in adhesion molecules in neurons. Biochemical studies have demonstrated the presence of binding sites for the Pax6 transcription factor in the NCAM and L1 promoter. Thus, increased proliferation or adhesion among neurons in the clusters could prevent Sey mutant cells from detaching from each other to reach their final position in the cerebral cortex (Morante, 2011).

Therefore, development of medulla neurons of the optic lobe resembles the neurogenesis and migration mechanisms observed during the development of the embryonic mammalian brain (Morante, 2011).

raw Functions through JNK signaling and cadherin-based adhesion to regulate Drosophila gonad morphogenesis

To form a gonad, germ cells (GCs) and somatic gonadal precursor cells (SGPs) must migrate to the correct location in the developing embryo and establish the cell-cell interactions necessary to create proper gonad architecture. During gonad morphogenesis, SGPs send out cellular extensions to ensheath the individual GCs and promote their development. Mutations have been identified in the raw gene that result in a failure of the SGPs to ensheath the GCs, leading to defects in GC development. Using genetic analysis and gene expression studies, it was found that Raw negatively regulates JNK signaling during gonad morphogenesis, and increased JNK signaling is sufficient to cause ensheathment defects. In particular, Raw functions upstream of the Drosophila Jun-related transcription factor to regulate its subcellular localization. Since JNK signaling regulates cell adhesion during the morphogenesis of many tissues, the relationship was examined between raw and the genes encoding Drosophila E-cadherin and beta-catenin, which function together in cell adhesion. It was found that loss of DE-cadherin strongly enhances the raw mutant gonad phenotype, while increasing DE-cadherin function rescues this phenotype. Further, loss of raw results in mislocalization of beta-catenin away from the cell surface. Therefore, cadherin-based cell adhesion, likely at the level of beta-catenin, is a primary mechanism by which Raw regulates germline-soma interaction (Jemc, 2012).

raw is an important regulator of embryonic gonad morphogenesis and the establishment of proper gonad architecture. raw mutants exhibit a failure of SGPs to ensheath GCs in the gonad, resulting in defects in GC development. It was also found that raw affects gonad morphogenesis primarily by acting as a negative regulator of the JNK signaling pathway. Finally, it was found that raw mutants exhibit defects in cadherin-based cell adhesion, and that this is the primary cause of the failure of gonad morphogenesis. These results have clear implications for understanding of how important cell signaling pathways are regulated to control normal organogenesis and may be misregulated to cause disease (Jemc, 2012).

Previously raw has been proposed to be a negative regulator of the JNK pathway during closure of the dorsal epidermis, based on changes in JNK-dependent expression of target genes such as dpp and puc. Indeed, an increase was seen in puc expression in the region of the embryonic gonad, and more broadly throughout the embryo. Further, upregulation of a dedicated AP-1 reporter construct was observed, indicating that the changes in target gene expression are directly due to changes in AP-1 transcriptional activity regulated by the JNK pathway. When the JNK pathway was upregulated via independent means, similar defects were observed in gonad morphogenesis, indicating that the changes in the JNK pathway were the primary mechanism by which raw mutants cause gonad defects. Therefore, the results support and extend the previous observations that raw acts as a negative regulator of JNK pathway, both in the gonad and in other tissues in which raw mutants exhibit defects in morphogenesis (Jemc, 2012).

How might raw be regulating the JNK pathway? The evidence indicates that raw regulates the JNK pathway at the level of transcription factor JRA. It was found that the nuclear localization of JRA, but not FOS, was altered in raw mutants. JRA was more strongly concentrated in the nucleus in a variety of cell types in raw mutants, whereas no changes were observed in the global levels of JRA protein. These observations are consistent with previous genetic epistasis experiments that indicated that raw acts at the level of JRA, rather than further upstream in the pathway. It has been proposed that raw acts as a general negative regulator of the JNK pathway to suppress basal activity and perhaps establish a threshold for pathway activation. The data are consistent with this hypothesis, as a general nuclear accumulation of JRA was seen in a variety of cell types in the embryo, along with generalized activation of the transcriptional reporter for AP-1 activity. Presumably, not all of these different cells are normally exposed to activators of the JNK pathway at this time, indicating that the pathway may be activated in cells in which the pathway would normally be turned off. Thus, rather than being just a modulator of the level of signal a cell might receive under conditions of JNK pathway activation, raw is likely also responsible for ensuring that the pathway remains inactive in cells that are not experiencing pathway activation (Jemc, 2012).

It is difficult to predict exactly how Raw may be regulating JNK signaling, as the Raw protein has no readily identifiable protein domains and exhibits only limited homology to proteins of other species. It may be the case that similar JNK pathway regulators are present in other species and have structural and/or functional conservation with Raw, but are difficult to identify based on primary sequence homology. Studies examining the subcellular localization of Raw in cultured mammalian or Drosophila cells indicate that it is primarily found in the cytoplasm. One attractive hypothesis is that Raw directly binds to JRA to block its nuclear translocation and sequester JRA in the cytoplasm. Unfortunately, efforts to identify a direct, physical interaction between Raw and JRA have so far been unsuccessful (Jemc, 2012).

The JNK pathway is subject to negative regulation at several levels. Most familiar are the MAP kinase phosphatases (MKPs, a subfamily of Dual-specificity phosphatases), like Drosophila Puckered, that provide negative feedback by dephosphorylating activated MAP kinases such as JNK. Additional modes of regulation include nuclear repressors of AP-1 target genes (e.g., Anterior open) and a secreted protease that acts in negative feedback on the JNK pathway (Scarface). Raw appears to represent a distinct mode of regulation, acting on the ability of JRA/JUN to translocate to the nucleus. Regulation of the subcellular localization of transcription factors and cofactors is a strategy that is commonly deployed to regulate signaling pathway activity, and many transcription factors are sequestered in the cytoplasm as a mechanism for negatively regulating their activity. It is proposed that JRA is subject to such regulation as a means to repress its activity in cells that are not experiencing sufficient levels of JNK pathway activity (Jemc, 2012).

Further studies of Raw are necessary to determine how Raw functions at a molecular level to regulate JRA subcellular localization (Jemc, 2012).

This study has found that raw mutants also exhibit defects in cadherin-based cell adhesion, which is known to be important for proper gonad morphogenesis and GC ensheathment by SGPs. Loss of DE-cad function exacerbates the gonad defects observed in raw mutants while increasing DE-cad function strikingly rescues these defects. It is likely that the increase in JNK pathway activity in raw mutants leads to defects in DE-cad-based adhesion and that this is the primary cause of the gonad morphogenesis defects. This is in contrast to the role of raw and the JNK pathway in the closure of the dorsal epidermis, which is largely thought to be due to regulation of dpp expression. Consistent with this, less up-regulation of dpp was observed in the region of the gonad, relative to the overall activation of the AP-1 transcriptional reporter (Jemc, 2012).

Previous studies in mammalian cells have implicated JNK signaling in negative regulation of cadherin-based cell adhesion, while in other contexts the JNK pathway has also been observed to upregulate DE-cad. The current results favor a repressive role for the JNK pathway on DE-cad in the gonad. It is also known that cadherins can act upstream of the JNK pathway, and that loss of cadherin can lead to an increase in c-Jun protein levels. However, the current results are consistent with DE-cad acting downstream of the JNK pathway, since DE-cadherin expression could rescue gonad morphogenesis independently of rescuing JRA localization. It is concluded that during gonad morphogenesis, raw acts as a negative regulator of the JNK pathway, and increased JNK pathway activity observed in raw mutants leads to a downregulation of DE-cadherin based cell adhesion and a failure of proper ensheathment of the GCs by the somatic gonad (Jemc, 2012).

While no change was observed in DE-cad localization in the gonad, the localization of ARM/ß-catenin was dramatically altered. Since ARM is essential for proper DE-cad function in cell adhesion, this indicates that DE-cadherin-based adhesion is strongly affected in raw mutants. It has been shown that JNK can directly phosphorylate ß-catenin and negatively regulate its activity. Consistent with this, a modest increase was observed in the relevant phospho-form of ARM/ß-catenin in raw mutants. Thus, this may represent one aspect of how the JNK pathway regulates DE-cad based adhesion in the gonad. However, the change in ARM/ß-catenin phosphorylation observed is unlikely to account for the more dramatic change in ARM/ß-catenin immunostaining observed in the gonad. Considering that a strong increase was also observed in transcriptional activation by AP-1 in raw mutants, and that mutations in the JRA transcription factor can partially suppress the gonad morphogenesis defects observed in raw mutants, it is concluded that at least some of the JNK pathway effect on DE-cad function and ß-catenin localization is likely to depend on changes in gene expression mediated by AP-1. Since no overall changes were observed in protein levels for DE-cad or ARM/ß-catenin in raw mutants, the changes in gene expression may reflect changes in other regulators of DE-cad based cell adhesion. Interestingly, previous work identified a zinc transporter, Fear of intimacy, that also affects gonad morphogenesis and GC ensheathment by regulating DE-cad. Regardless of whether there is an interesting connection between zinc transport and the JNK pathway, or these represent independent pathways, they highlight the importance of careful regulation of cadherin-based cell adhesion in controlling morphogenesis (Jemc, 2012).

Previous work has indicated that GC ensheathment requires preferential adhesion between SGPs and GCs, such that SGP-GC adhesion is favored over GC-GC or SGP-SGP adhesion. Indeed, just increasing the adhesion between GCs via DE-cadherin expression in these cells is sufficient to prevent ensheathment of the GCs by SGPs. In raw mutants, changes were primarily observed in the JNK pathway in SGPs and surrounding somatic cells. In addition, expression of DE-cad in the soma, but not the germline, is sufficient to rescue the ensheathment defects in raw mutants. Together, these data indicate that raw mutants likely affect gonad ensheathment by decreasing DE-cad function in the SGPs, which decreases SGP-GC adhesion relative to GC-GC adhesion. While it is possible that effects of raw on somatic cells outside of the gonad affect ensheathment within the gonad, it is less easy to imagine how decreasing DE-cad activity in these cells would influence ensheathment (Jemc, 2012).

The JNK pathway has been implicated in many diseases, including birth defects, neurodegeneration, inflammatory diseases, and cancer. Signaling pathways must be tightly regulated both positively, to ensure rapid and robust signaling responses, and negatively, to terminate signaling events and prevent inappropriate signaling. As a negative regulator of JNK pathway signaling, raw represents the type of gene that might be mutated or misregulated in diseases caused by altered JNK pathway activity. This idea is supported by the strong developmental phenotypes associated with mutations in negative regulators of the JNK pathway in Drosophila and mice (Jemc, 2012).

One disease where the JNK pathway has been particularly well studied is cancer. The JNK pathway's role in cancer is complex, however, and the pathway can act in tumor suppression or oncogenesis, depending on the context. In mouse and Drosophila models of cancer due to activated Ras, upregulation of the JNK pathway is required for tumor formation and disease progression. Interestingly, downregulation of E-cadherin is also associated with cancer progression, including in the models of activated Ras where the JNK pathway is involved. Thus, a similar link between the JNK pathway and cadherin regulation that was observed in morphogenesis of the gonad during development may play a role in oncogenesis. Since upregulation of the JNK pathway promotes cancer in these examples, negative regulators of the pathway such as the MAPK phosphatases or Raw would act as tumor suppressors whose mutation could contribute to disease progression. A better understanding of how the JNK pathway is regulated, and how Raw contributes to this regulation, is essential for understanding the normal roles of the JNK pathway in development and homeostasis, and how it is misregulated to cause disease (Jemc, 2012).

E-cadherin-mediated cell coupling is required for apoptotic cell extrusion

Apoptotic extrusion is a multicellular process utilized by live cells to remove neighboring apoptotic cells. In epithelial tissues, this process has been shown to be critical for the preservation of tissue integrity and barrier function. This study demonstrates that extrusion is driven by the retraction of the apoptotic cell, which, in turn, triggers a transient and coordinated elongation of the neighboring cells. The coordination of cell elongation requires E-cadherin-mediated cell-cell adhesion. Accordingly, cells that express low levels of E-cadherin are compromised in elongation and apoptotic extrusion, and furthermore, display loss of barrier function in response to apoptotic stimuli. These findings indicate that the maintenance of adhesive forces during apoptotic cell turnover might play an essential role in controlling tissue homeostasis (Lubkov, 2014).

Differential regulation of the Hippo pathway by adherens junctions and apical-basal cell polarity modules

Adherens junctions (AJs) and cell polarity complexes are key players in the establishment and maintenance of apical-basal cell polarity. Loss of AJs or basolateral polarity components promotes tumor formation and metastasis. Recent studies in vertebrate models show that loss of AJs or loss of the basolateral component Scribble (Scrib) cause deregulation of the Hippo tumor suppressor pathway and hyperactivation of its downstream effectors Yes-associated protein (YAP) and Transcriptional coactivator with PDZ-binding motif (TAZ), homologs of Drosophila Yorkie. However, whether AJs and Scrib act through the same or independent mechanisms to regulate Hippo pathway activity is not known. This study dissects how disruption of AJs or loss of basolateral components affect the activity of the Drosophila YAP homolog Yorkie (Yki) during imaginal disc development. Surprisingly, disruption of AJs and loss of basolateral proteins produced very different effects on Yki activity. Yki activity was cell-autonomously decreased but non-cell-autonomously elevated in tissues where the AJ components E-cadherin (E-cad) or α catenin (α-cat) were knocked down. In contrast, scrib knockdown caused a predominantly cell-autonomous activation of Yki. Moreover, disruption of AJs or basolateral proteins had different effects on cell polarity and tissue size. Simultaneous knockdown of α-cat and scrib induced both cell-autonomous and non-cell-autonomous Yki activity. In mammalian cells, knockdown of E-cad or α-cat caused nuclear accumulation and activation of YAP without overt effects on Scrib localization and vice versa. Therefore, these results indicate the existence of multiple, genetically separable inputs from AJs and cell polarity complexes into Yki/YAP regulation. (Yang, 2014).

This report addresses the effects of AJs and basolateral cell polarity determinants on the activity of the Hippo pathway in Drosophila imaginal discs. Knockdown of AJs and basolateral components both induced ectopic activation of Yki. However, knockdown of AJs and basolateral proteins had strikingly different effects on Yki. Disruption of the basolateral module induced mainly a cell-autonomous increase in Yki activity, whereas knockdown of AJs caused non-autonomous induction of Yki reporters. Therefore, these data identify and genetically uncouple multiple different molecular pathways from AJs and the basolateral module that regulate Yki activity (Yang, 2014).

These studies further show that knockdown of AJs induces cell-autonomous reduction of Yki activity and causes cell death and decreased size of Drosophila imaginal discs. Likewise, E-cad and :alpha;-cat mutant clones do not survive in imaginal discs. This effect may be mediated by LIM domain proteins of the Zyxin and Ajuba subfamilies, which regulate Hippo signaling by directly inhibiting Wts/Lats kinases and by interacting with Salvador (Sav), an adaptor protein that binds to the Hpo/MST kinases. A recent report shows that α-Cat recruits Ajuba and indirectly Wts to AJs and loss of Ajuba leads to activation of Wts and hence phosphorylation and inhibition of Yki and diminished tissue size. Thus, α-cat mutant cells may inactivate Yki because they lose Ajuba function (Yang, 2014).

In contrast, in mammalian systems, several in vivo and in vitro studies have shown the opposite effect on Hippo signaling upon AJ disruption; knockdown of E-cad or α-cat caused an increase in cell proliferation and nuclear accumulation of YAP, and conditional knockout of α-cat in mouse skin cells caused tumor formation and elevated nuclear YAP staining. This suggests that AJ components have a tumor suppressor function in mammals. The observation that Scrib is mislocalized upon disruption of AJs in several different mammalian cell lines suggested that YAP activation could be due to the concomitant disruption of the basolateral module. However, the finding that acute disruption of AJs can cause YAP activation without disrupting Scrib localization and vice versa indicates that AJs and the basolateral module also act independently on the Hippo pathway in mammalian cells. In mammalian cells, α-Cat forms a complex with YAP and 14-3-3 proteins, thereby sequestering phosphorylated YAP at the plasma membrane. However, α-Cat may function as a tumor suppressor only in epidermal stem cells, as conditional deletion of α-cat in differentiated cells only caused a mild phenotype with no overgrowth and tumor formation. Therefore, it is possible that the negative regulation of YAP by α-Cat is cell type-specific, although further testing is required to fully address this issue (Yang, 2014).

The non-cell-autonomous effect of AJ knockdown on the Hippo pathway is an intriguing phenomenon. Several groups reported non-autonomous effects on the Hippo pathway in Drosophila in other mutant conditions. Disrupting the expression gradients of the atypical Cadherin Dachsous or that of its regulator Four-jointed, clones of cells mutant for the tumor suppressor genes vps25 or hyperplastic discs (hyd) , clones of cells overexpressing Src64, or overexpression of the proapoptotic gene reaper or the JNK signaling ligand eiger all cause non-autonomous activation of Yki. This non-autonomous activation of Yki may be part of a regenerative response that stimulates cell proliferation in cells neighboring tissue defects. The signals that activate Yki in these situations are not known, nor is it known whether these mutant conditions activate the same or different signaling mechanisms. The non-autonomous activation of Yki around cells with AJ knockdown may be mediated by changes in mechanical forces. AJs are important for maintaining tension between cells across epithelia, and disruption of AJs leads to an imbalance of apical tension. Mechanical forces are known to regulate the Hippo pathway, and YAP/TAZ act as mediators of mechanical cues from the cellular microenvironment such as matrix stiffness. In particular, the Zyxin and Ajuba family LIM domain proteins can act as sensors of mechanical forces and may be involved in the non-autonomous activation of Yki. The effects on Hippo signaling of solely changing Zyxin and Ajuba may not be as strong as those described in this study, and these proteins may thus cooperate with other molecular conduits to regulate the activity of the Hippo pathway in response to changes in AJ strength. Unraveling these mechanisms will provide important new insights into understanding how cells interact with neighboring cells to regulate proliferation, apoptosis, and the Hippo pathway (Yang, 2014).

It is currently unknown whether AJs also exert non-autonomous effects on the Hippo pathway in mammalian tissues. Amphiregulin, an EGF ligand, is a downstream target of YAP and can induce non-cell-autonomous cell proliferation through EGFR signaling. However, it is not known whether YAP itself is activated non-cell-autonomously to contribute to the hyper-proliferation phenotypes observed upon disruption of AJs in vivo and in vitro. It will be interesting to determine whether AJs and other cell-cell signaling mechanisms also have non-cell-autonomous effects on the activity of YAP in mammalian tissues, for example during regeneration (Yang, 2014).

Finally, the apical proteins aPKC and Crb modulate the activity of the Hippo pathway, and many Hippo pathway components are apically localized, which is important for their activity. The data presented in this study add to these findings. Disruption of AJs causes reduced Yki activity, despite the fact that Crb and Mer are mislocalized. Thus, AJs and cell polarity components regulate Yki activity through multiple, genetically separable inputs. It will be interesting to decipher all of the different underlying molecular mechanisms of how AJs and basolateral proteins regulate the Hippo pathway and how these mechanisms evolved in Drosophila and in mammals (Yang, 2014).

A role for E-cadherin in ensuring cohesive migration of a heterogeneous population of non-epithelial cells

Collective cell migration is a key process underlying the morphogenesis of many organs as well as tumour invasion, which very often involves heterogeneous cell populations. This study investigates how such populations can migrate cohesively in the Drosophila posterior midgut, comprised of epithelial and mesenchymal cells and shows a novel role for the epithelial adhesion molecule E-cadherin (E-Cad) in mesenchymal cells. Despite a lack of junctions at the ultrastructure level, reducing E-Cad levels causes mesenchymal cells to detach from one another and from neighbouring epithelial cells; as a result, coordination between the two populations is lost. Moreover, Bazooka and recycling mechanisms are also required for E-Cad accumulation in mesenchymal cells. These results indicate an active role for E-Cad in mediating cohesive and ordered migration of non-epithelial cells, and discount the notion of E-Cad as just an epithelial feature that has to be switched off to enable migration of mesenchymal cells (Campbell, 2015).

Drosophila E-cadherin is required for the maintenance of ring canals anchoring to mechanically withstand tissue growth

Intercellular bridges called "ring canals" (RCs) resulting from incomplete cytokinesis play an essential role in intercellular communication in somatic and germinal tissues. During Drosophila oogenesis, RCs connect the maturing oocyte to nurse cells supporting its growth. Despite numerous genetic screens aimed at identifying genes involved in RC biogenesis and maturation, how RCs anchor to the plasma membrane (PM) throughout development remains unexplained. This study reports that the clathrin adaptor protein 1 (AP-1) complex, although dispensable for the biogenesis of RCs, is required for the maintenance of the anchorage of RCs to the PM to withstand the increased membrane tension associated with the exponential tissue growth at the onset of vitellogenesis. It was shown that AP-1 regulates the localization of the intercellular adhesion molecule E-cadherin and that loss of AP-1 causes the disappearance of the E-cadherin-containing adhesive clusters surrounding the RCs. E-cadherin itself is shown to be required for the maintenance of the RCs' anchorage, a function previously unrecognized because of functional compensation by N-cadherin. Scanning block-face EM combined with transmission EM analyses reveals the presence of interdigitated, actin- and Moesin-positive, microvilli-like structures wrapping the RCs. Thus, by modulating E-cadherin trafficking, it was shown that the sustained E-cadherin-dependent adhesion organizes the microvilli meshwork and ensures the proper attachment of RCs to the PM, thereby counteracting the increasing membrane tension induced by exponential tissue growth (Loyer, 2015).

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