Analysis of the spatio-temporal expression of the l(2)gl transcripts and proteins has shown that transcripts and proteins are produced ubiquitously during early embryogenesis, at a time when expression of the gene is required for preventing tumorigenesis. In the second half of embryogenesis, l(2)gl expression becomes restricted to tissues that do not show any phenotypic alteration in mutant animals. The l(2)gl protein exhibits two distinct intracellular localizations. It is preferentially found free in the cytoplasm but can become associated with the inner face of the plasma membrane where it is restricted to domains facing contiguous cells. In particular, the l(2)gl protein is absent from the basal and apical domains of the plasma membrane (Strand, 1991).
The relation between scrib, lgl, and dlg were explored by comparing the subcellular localization of the gene products. Scrib and Dlg colocalize throughout development, in particular at the apical margin of the lateral membrane (ALM) of the embryonic epidermal epithelium. Scrib is localized to the epithelial septate junction, the analog of the vertebrate tight junction, at the boundary of the apical and basolateral cell surfaces. Colocalization at the ALM occurs after gastrulation and persists in mature epithelia, where the ALM is the site of the septate junction. Lgl protein is not exclusively associated with the plasma membrane and is not polarized along it; however, it overlaps substantially with Dlg and Scrib at the ALM (Bilder, 2000b).
Asymmetric cell division is important in generating cell diversity from bacteria to mammals. Drosophila neuroblasts are a useful model system for investigating asymmetric cell division because they establish distinct apical-basal cortical domains, have an asymmetric mitotic spindle aligned along the apical-basal axis, and divide unequally to produce a large apical neuroblast and a small basal daughter cell (GMC). Discs large (Dlg), Scribble (Scrib) and Lethal giant larvae (Lgl) tumour suppressor proteins regulate multiple aspects of neuroblast asymmetric cell division. Dlg/Scrib/Lgl proteins show apical cortical enrichment at prophase/metaphase, and then have a uniform cortical distribution. Mutants have defects in basal protein targeting, a reduced apical cortical domain and reduced apical spindle size. Defects in apical cell and spindle pole size result in symmetric or inverted neuroblast cell divisions. Inverted divisions correlate with the appearance of abnormally small neuroblasts and large GMCs, showing that neuroblast/GMC identity is more tightly linked to cortical determinants than cell size. It is concluded that Dlg/Scrib/Lgl are important in regulating cortical polarity, cell size asymmetry and mitotic spindle asymmetry in Drosophila neuroblasts (Albertson, 2003).
Little is known about how intercellular communication is regulated in epithelial cell clusters to control delamination and migration. This problem has been investigated using Drosophila border cells as a model. Just preceding cell cluster delamination, expression of transmembrane immunoglobulin superfamily member, Fasciclin 2, is lost in outer border cells, but not in inner polar cells (PCs) of the cluster. Loss of Fasciclin 2 expression in outer border cells permits a switch in Fasciclin 2 polarity in the inner polar cells. This polarity switch, which is organized in collaboration with neoplastic tumor suppressors Discs large and Lethal-giant-larvae, directs cluster asymmetry essential for timing delamination from the epithelium. Fas2-mediated communication between polar and border cells maintains localization of Discs large and Lethal-giant-larvae in border cells to inhibit the rate of cluster migration. These findings are the first to show how a switch in cell adhesion molecule polarity regulates asymmetry and delamination of an epithelial cell cluster. The finding that Discs large and Lethal-giant-larvae inhibit the rate of normal cell cluster movement suggests that their loss in metastatic tumors may directly contribute to tumor motility. Furthermore, these results provide novel insight into the intimate link between epithelial polarity and acquisition of motile polarity that has important implications for development of invasive carcinomas (Szafranski, 2004).
How does developmentally programmed loss of Fas2 expression in BCs permit Fas2 polarization in PCs? The data indicate that this is a multistep process. Initially, Fas2 homophilic interactions between BCs and PCs are lost, and several experiments indicate that they are replaced by Fas2 heterophilic interactions with a putative BC receptor. These interactions are essential for maintaining Fas2 in PC membranes contacting BCs. Next, loss of Fas2 from BCs causes relocation of the majority of PC Fas2 to the interface between PCs, where it is maintained because of homophilic interactions with Fas2 from the adjacent PC. In support of this interpretation, misexpression of Fas2 in PCs appears to oversaturate Fas2 between PCs, causing its circumferential accumulation at the contact sites with BCs. It is concluded that the accumulation of Fas2 between PCs ensures that Fas2 is kept at sufficiently low levels at the sites of contact with BCs to allow its polarization to the leading half of PCs. Fas2 polarization is then directed by PC Dlg and Lgl, as evidenced by the observation that loss of function of either protein causes loss of Fas2 polarity. However, Fas2 can also polarize Dlg and Lgl; loss of Fas2 causes loss of Dlg and Lgl polarity, while ectopic Fas2 redirects Dlg and Lgl localization. Thus, Fas2 is in a positive feedback loop with Dlg and Lgl that ensures the build up of a PC signaling and adhesion complex at the leading half of the PCs. These data indicate that Fas2 is involved in intercellular interactions crucial for organizing polarity, an important criterion for a function specifically involved in regulation of motility in multicellular clusters (Szafranski, 2004).
Significantly, the results indicate that molecules used for polarizing epithelial cells are reorganized to polarize a motile cell cluster. The timing of the reorganization of epithelial polarity is crucial for timing delamination. Fas2 therefore plays a direct role in mediating intercellular interactions that modulate movement, a second property proposed for a function specifically involved in regulating cluster motility as opposed to single cells. It is concluded that the Fas2 morphogentic switch facilitates development of motile polarity essential for timely BC delamination. A similar switch mechanism may be important in other processes that crucially depend on timing of Fas2 activity, such as axon pathfinding, and learning and memory (Szafranski, 2004).
Fas2 polarity appears to compartmentalize PCs into distinct functional domains in order to control functionally distinct intercellular communication with leading versus trailing BCs. Leading BCs play a functionally distinct role by pioneering invasion between germ cells while simultaneously detaching from the epithelium. Trailing BCs are likely to play a less active role in invasion, but must mediate precisely timed detachment from the epithelium. Fas2 polarization is thus likely to be crucial for facilitating coordination of the distinct functional requirements of leading versus trailing BCs, by establishing distinct sets of intercellular contact and communication between the PCs and leading versus trailing BCs. In support of this hypothesis, previous studies have suggested that leading and trailing BCs are functionally distinct. In BC clusters comprising a mixture of wild type and slbo, jing, taiman or DE-cadherin mutant cells, wild-type BCs always lead invasion. Furthermore, additional structural evidence has been documented for cluster asymmetry. Amphiphysin, a vesicle trafficking protein that regulates Dlg and Lgl localization, is expressed at higher level in trailing BCs compared to leading BCs. Amph, Dlg and Lgl, are thus good candidates for proteins that differentially regulate cortical and cell surface activities needed to mediate distinct interactions of leading and trailing BCs with adjacent epithelial cells and germ cells during the delamination process (Szafranski, 2004).
Since only Dlg and Lgl are mislocalized in Fas2 clusters, but not Fas3, alpha-Spec or Crb, the data suggest that Fas2 directs localization of specific molecules within distinct regions of different cells of the cluster to control motility. A putative Fas2-binding BC receptor may be another molecule whose polarity is controlled by Fas2. Interaction with this putative receptor appears to facilitate organization of the global polarity of the cluster, since the orientation of delamination, mediated by the BCs, directly correlates with Fas2 polarity in PCs. These data thus suggest that Fas2 coordinates directional mass motion between cells that are potentially capable of motion in any direction, and that it helps to determine the locomotive-active regions of these cells, additional criteria for a function specifically involved in regulating cluster motility. Thus, because Fas2 is required for regulation of several activities that distinguish how single cells versus clusters move, the data provide the first molecular model for understanding the organization of epithelial cluster polarity during delamination and movement. One argument against this proposal might be that the PCs appear to be highly specialized. However, it is thought that this is likely to be of less significance, since PCs express epithelial polarity proteins in a pattern similar to adjacent follicle epithelial cells (Szafranski, 2004).
As has been shown for BC clusters, several vertebrate studies have shown that transmembrane proteins are differently expressed within different cell subpopulations in migrating clusters. Furthermore, the structure and functions of Fas2, Dlg and Lgl homologs are conserved across phylogeny. Thus, the involvement of Fas2, Dlg and Lgl in organizing cell cluster motility also may be conserved. It is concluded that although the precise mechanism of cluster movement may not be conserved in vertebrates, the information gleaned about how BCs regulate epithelial polarity to dynamically organize cluster polarity and movement will be generally useful for understanding how cell cluster motility is organized across phylogeny (Szafranski, 2004).
The role of Fas2 in regulating migration is discussed. Loss- and gain-of-function experiments demonstrate that PC Fas2 acts as a signal to inhibit the rate of BC migration. This work builds on previous studies demonstrating the importance of PCs in determining BC fate. However, this work is the first example of an intercellular signal that specifically organizes cluster movement, rather than determining cell fate. Fas2 clearly has a signaling function, since PCs do not contact the migration substrate. Thus, these data demonstrate for the first time the existence of intercellular communication between cells of a migratory cluster, that is specifically required to modulate migration (Szafranski, 2004).
PC Fas2 signaling inhibits the rate of cluster movement by maintaining Dlg and Lgl localization in BCs. The putative BC receptor with which Fas2 interacts may control Dlg and Lgl localization in BCs. Since Dlg is localized to the cortex of BCs, Dlg must inhibit the rate of migration through cortical activities in BCs. One cortical activity controlled by Dlg is the recruitment of Lgl to the membrane. Since lgl clusters have very similar migration phenotypes to dlg clusters, the data indicate that Lgl and Dlg cooperate to inhibit BC movement. The importance of Dlg and Lgl in regulating cell movement probably derives from the same scaffolding activities they use to organize and control membrane, cytoskeletal and signaling specialization during the polarization of epithelial and neuronal cells. It is proposed that Dlg and Lgl scaffolding organizes and integrates transmembrane signaling and adhesion proteins with signaling, trafficking and cytoskeletal effectors in the cortex of BCs to mediate contact-inhibition of cluster movement (Szafranski, 2004).
BCs resemble mutant dlg invasive tumor cells in that they lose epithelial polarity by accumulating Dlg and Lgl around their circumference, but in contrast to BCs, mutant dlg tumor cells migrate between germ cells without temporal or spatial control. The data demonstrate that Dlg and Lgl not only control polarity and delamination of epithelial clusters, but also actively inhibit movement. Thus, mutant dlg tumor invasion is likely to be caused by a combination of loss of epithelial polarity and over-activation of motility pathways. In this context the results appear to be paradoxical in that loss of epithelial polarity is generally considered to be crucial for facilitating acquisition of motility, but it is seen that loss of polarity in normal migrating clusters delays initiation of movement. The data resolve this paradox in that during normal development, molecules used for polarizing epithelial cells are reorganized to polarize a motile cell cluster. It therefore seems likely that in carcinomas, inappropriate loss of epithelial polarity simultaneously disrupts acquisition of motile polarity, but this phenomenon is not appreciated because ultimately the tumor cells migrate. Thus, it is postulated that overactivation of motility pathways, as is seen with loss of Dlg and Lgl in BCs, may be especially crucial for achieving carcinoma invasion. Consistent with this hypothesis, some dlg mutations that cause loss of epithelial polarity do not lead to tumor invasion, suggesting that acquisition of motility is a separate Dlg function (Szafranski, 2004).
Gene expression data for human cancers suggests that mutations that promote tumor formation, through loss of epithelial polarity and increased proliferation, may be the same mutations that subsequently cause tumor cell invasion. Based on the observation that Dlg is required to maintain polarity, inhibit proliferation and inhibit movement, it is proposed that tumor suppressors such as Dlg that regulate signaling and adhesion at epithelial junctions may unify human gene expression data by providing an ultrastructural target that controls contact inhibition of both proliferation and movement. Progressive deterioration of epithelial junctions may thus provide a common mechanism through which multiple tumor suppressor pathways impact the cascade from cell proliferation to tumor invasion, either through mutation or mislocalization of critical junctional proteins (Szafranski, 2004).
Loss of cell polarity and tissue architecture are characteristics of malignant cancers derived from epithelial tissues. Cells in epithelial sheets are characterized by columnar or cuboidal shape, strong cell-cell adhesion, and pronounced apicobasal polarity. However, tumors of epithelial origin lose these characteristics as they progress from benign growth to malignant carcinoma, and this loss is associated with poor clinical prognosis. Evidence is provided that a group of membrane-associated proteins act in concert to regulate both epithelial structure and cell proliferation. Scribbled (Scrib) is a cell junction-localized protein required for polarization of embryonic and imaginal disc and follicular epithelia. The tumor suppressor scrib was isolated in a screen for maternal effect mutations that disrupt aspects of epithelial morphogenesis such as cell adhesion, shape and polarity. scrib encodes a multi-PDZ (PSD-95, Discs-large and ZO-1) and leucine-rich-repeat protein. The structure of the embryonic cuticle was used to reflect the organization of the underlying epithelial epidermis that secretes it. The wild-type cuticle forms a smooth, continuous sheet, but embryos that are maternally and zygotically mutant for scrib produce a corrugated cuticular surface that is riddled with holes, hence the name scribbled (Bilder, 2000a).
Two other tumor suppressors, lethal giant larvae (lgl) and discs-large (dlg), have the identical effects as scrib mutation on epithelial structure. Scrib and Dlg colocalize and overlap with Lgl in epithelia; activity of all three genes is required for cortical localization of Lgl and junctional localization of Scrib and Dlg. scrib, dlg, and lgl show strong genetic interactions. Thus, these three tumor suppressors act together in a common pathway to regulate cell polarity and growth control (Bilder, 2000b).
The equivalent requirements for scribbled, lgl, and dlg could result from independent activity of each gene in a separate pathway or from collaborative activity of the three genes in a single pathway. To address this issue, genetic interactions between the three mutations were tested and strong interactions of dlg and lgl with scrib were found. Most embryos zygotically mutant for scrib hatch and survive into late larval stages. However, embryos homozygous for scrib and additionally heterozygous for dlg die before hatching, with evident defects in dorsal closure. Dorsal closure phenotypes are characteristic of reduced activity of both dlg and lgl. Additionally, embryos homozygous for both lgl and scrib display a cuticle phenotype nearly as severe as those of lgl or scrib null embryos. Heterozygosity for lgl also enhances the imaginal disc phenotype of scrib hypomorphic larvae. These dose-sensitive interactions of dlg and lgl with scrib suggest that the three genes function in a common pathway (Bilder, 2000b).
Epistatic relations between scrib, lgl, and dlg were investigated by determining the localization of each protein in embryos mutant for the other two genes. The localization of Dlg and Scrib to the ALM was examined. In all mutant blastoderms, Scrib and Dlg are associated with ingrowing cell membranes, as in WT. However, after gastrulation, when WT embryos display an enrichment of Scrib in the ALM, lgl embryos show Scrib and Dlg localized throughout the basolateral cell membrane, a misdistribution that persists into late embryogenesis. Like lgl embryos, scrib embryos fail to polarize Dlg to the ALM, while dlg embryos not only fail to polarize Scrib to the ALM, but also display a progressive loss of membrane-associated Scrib (Bilder, 2000b).
Also examined was the distribution of Lgl, which normally has both a membrane-bound and a cytosolic component; reduction of activity in an lgl temperature-sensitive mutant correlates with loss of the membrane-bound pool. In WT embryos, Lgl is in close apposition to cell membranes. However, in scrib blastoderms and embryos, Lgl is distributed throughout the cytoplasm. dlg blastoderms show intermediate defects in Lgl distribution, but by mid-embryogenesis loss of membrane-localized Lgl is evident. The dissociation of Lgl from the membranes of dlg embryos parallels the loss of Scrib seen in these embryos. These data indicate that dlg is required for the stable association of Scrib with the cell membrane and scrib is required for the cortical association of Lgl; all three genes act to localize Scrib and Dlg to the ALM (Bilder, 2000b).
To test whether the requirement for Scrib is limited to the embryo, an examination was made of the role of scrib in follicle cells, a monolayered epithelium of somatic cells that encases the germ line in the adult female ovary. Scrib is localized to lateral follicle cell membranes, and clones of cells that lack scrib function become round and multilayered with polarity defects, similar to the phenotype of embryonic epithelia lacking scrib function. The epithelial defects of scrib mutant follicle cells are cell autonomous. These data indicate that scrib is required within cells from multiple tissues for proper epithelial structure (Bilder, 2000b).
Because follicle cell epithelia require scrib, lgl, and dlg, the functions of lgl and dlg were examined in the embryonic epidermis, where scrib acts to restrict apical proteins and adherens junctions to their appropriate positions within the cell membrane (Bilder, 2000a). Embryos lacking both maternal and zygotic contributions of lgl and dlg, (hereafter referred to as lgl and dlg embryos) were stained with antibodies to polarized proteins and cellular junction components. During mid-embryogenesis, lgl and dlg embryos show defects in apicobasal polarity, revealed by aberrant distribution of the apical protein Crumbs (Crb) and disruption of adherens junctions. These defects are similar to those of scrib embryos; the terminal phenotypes of scrib, lgl, and dlg embryos, as indicated by cuticle deposition, are also nearly identical. Thus, lgl and dlg, like scrib, act to properly localize apical proteins and adherens junctions to organize epithelial architecture in embryos (Bilder, 2000b).
The similarity of mutant phenotypes in different epithelia suggests that the three proteins are components of the fundamental machinery that creates the distinctive architecture of epithelial cells and tissues. To test this assertion, the scrib phenotype was compared to that of lgl and dlg in a third major epithelium, the larval imaginal disc. Discs isolated from late third instar larvae zygotically mutant for scrib are profoundly disorganized and also massively overgrown. scrib discs contain 4.7 times as many cells as wild-type (WT) discs and consist of spherical masses of tightly packed cells, as opposed to the folded monolayer epithelium seen in WT larvae. The apical polarization of actin evident in WT discs is absent in scrib discs. This loss of epithelial organization accompanied by overproliferation corresponds to the phenotype described for lgl and dlg zygotic mutant discs. Additional features of lgl and dlg larval phenotypes, such as overgrowth of brain tissue, are also present in scrib larvae. Together, these data indicate that scrib and the two previously characterized Drosophila malignant neoplastic tumor suppressors, lgl and dlg, share a role in growth control as well as epithelial polarity. Epistatic relations between scrib, lgl, and dlg were investigated by determining the localization of each protein in embryos mutant for the other two genes. These experiments have shown that dlg is required for the stable association of Scrib with the cell membrane and scrib is required for the cortical association of Lgl; all three genes act to localize Scrib and Dlg to the apical margin of the lateral membrane (ALM) of the embryonic epidermal epithelium (Bilder, 2000b).
These results provide strong evidence that Scrib, Dlg, and Lgl act in a common pathway to regulate cell architecture and cell proliferation control. Of the ~50 Drosophila genes in which mutation gives rise to overproliferation, only scrib shares with dlg and lgl the concomitant loss of tissue organization, which groups the three together as malignant neoplastic tumor suppressors. Previous analyses have described a role for dlg and lgl in imaginal disc polarity; the demonstration in this work of genetic interactions with scrib and codependence for protein localization indicates a functional link between the three tumor suppressors. Furthermore, involvement of the tumor suppressors in embryonic epithelial polarity provides a well-studied context in which to understand their activities. These findings suggest that, in the WT gastrula, intrinsic, perhaps adhesion-based cues localize Dlg at the ALM; Dlg stabilizes Scrib at this position, and finally Scrib acts on the cortical cytoskeleton to bring Lgl to the membrane. The three proteins may then collaborate to maintain the proper distribution of polarized factors, including themselves (Bilder, 2000b).
The correlation between loss of membrane-associated Lgl in scrib and dlg mutants and defective cell polarity suggests models of action for this group of proteins. Whereas the PDZ domains of Scrib and Dlg are likely to bind to transmembrane proteins that organize the epithelial cell surface, the role of Lgl in polarity determination may derive from its function in targeted secretion of membrane proteins. Lgl homologs from humans and yeast can bind to plasma membrane t-SNARE proteins and promote the fusion of cargo-carrying vesicles with target membranes (Fujita, 1998; Lehman, 1999). In yeast undergoing polarized growth, the broadly distributed Lgl homologs function primarily at the bud tip, the site of the 'exocyst' complex required for vesicle trafficking and addition (TerBush, 1996). In vertebrate epithelia, exocyst components are found at the tight junction, a structure analogous to the septate junction where Dlg and Scrib localize. In Drosophila epithelia, recruitment of Lgl into the proximity of membrane t-SNAREs requires proper localization of Scrib and Dlg, thus potentially linking the transmembrane proteins that establish polarity to the protein-targeting system that preserves it (Bilder, 2000b and references therein).
In many epithelial-derived cancers, cytoarchitectural changes are hallmarks of oncogenic transformation. The disruption of epithelial architecture seen in scrib, dlg, and lgl animals could affect growth control by several mechanisms. Many growth factor receptors are polarized to a specific membrane domain, and mislocalization of such proteins may affect signaling pathways that maintain cells in a differentiated, nonproliferative state. Additionally, the aberrant cell-cell junctions formed in scrib, dlg, and lgl mutants could compromise contact inhibition. Finally, disruption of cell-cell contacts may release junction-localized signaling components, such as Arm or APC, that have been implicated in regulating cell proliferation; indeed, a human Dlg homolog has been shown to bind APC and associate with beta-catenin, the human homolog of Arm. Because the modes of action of Scrib, Dlg, and Lgl are likely to be conserved between vertebrates and invertebrates, investigation into a tumorigenic role for the multiple human homologs of these genes is warranted. Further analysis of the mechanisms by which Scrib, Dlg, and Lgl keep Drosophila cell growth in check will likely enhance an understanding of mammalian oncogenesis as well (Bilder, 2000b).
Recessive mutations at the l(2)gl locus of Drosophila cause a complex syndrome, which has as its most striking features the development of malignant neuroblastomas in the larval brain and tumors of the imaginal discs. A chromosomal segment containing the l(2)gl gene has been cloned. Within this segment a transcription unit has been localized that is structurally changed in all l(2)gl alleles examined. The developmental profile of expression of the two RNAs (6 and 4.5 kb) made by this transcription unit coincides with the two major terminal phases of cell proliferation in the developing fly, namely, early embryogenesis and late third instar larvae. Tumors are produced when both normal l(2)gl alleles are inactivated by deletion or insertional mutation. The normal function of the l(2)gl presumably controls the normal cell proliferation of the optic centers of the brain and the imaginal discs, as well as their post-mitotic differentiation (Mechler, 1985).
Homozygous mutations of the recessive oncogene lethal 2 giant larvae of Drosophila cause lethal neoplasms of the imaginal discs and the brain hemisphere. A 13-kb DNA segment spanning the l(2)gl+ locus has been inserted into P element vectors and used for P-mediated transformation. The P-l(2)gl+ transposons have been introduced into the germ line of heterozygous l(2)gl-/+ flies and were shown by backcrossing to fully rescue the homozygous l(2)gl deficient animals, which otherwise would have died of brain and imaginal disc neoplasms. Further genetic backcrossing with l(2)gl deficiencies characterized by deletions of increased sizes involving the left end of chromosome 2 indicated that a relatively large region of developmentally regulated DNA sequence adjacent to the l(2)gl gene is apparently not essential for the viability and fertility of the fly. These experiments indicate that all the genetic information specified by the l(2)gl+ gene is contained within this 13-kb DNA segment and demonstrate that the development of neuroblastomas and imaginal disc tumors results from the absence of l(2)gl function. When this function is restored, tumor development is completely suppressed (Opper, 1987).
Cell proliferation in Drosophila imaginal discs appears to be regulated by a disc-intrinsic mechanism involving local cell interactions that also control the formation of patterns of differentiation. This growth-control mechanism breaks down in animals homozygous for the mutation lethal (2) giant discs; such mutants remain as larvae for up to 9 days longer than normal. During this time cell proliferation continues in the imaginal discs as well as in the imaginal rings for the salivary glands, foregut, and hindgut, so that these tissues become greatly overgrown. When wild-type wing discs from mid-third instar larvae were removed and cultured for up to 28 days in wild-type female adult hosts, they grew and terminated growth at a cell number close to that which would be attained in situ by the time of pupariation. In contrast, wing discs from l(2)gd homozygotes grew rapidly and continuously when cultivated in wild-type hosts, reached an enormous size, and acquired abnormal folding patterns. Overgrowth of mutant imaginal rings also continued during culture of these tissues in wild-type hosts. It is concluded that overgrowth in this mutant is due to an autonomous defect in the imaginal primordia, which requires an extended larval period for its expression in situ (Bryant, 1985).
Homozygosity for lethal(2)giant larvae induces neoplasia of the imaginal discs. To explore the developmental capacities of lgl mutant cells, their growth and differentiation has been investigated in genetic mosaics. Adult wings mosaic for lgl display abnormal growth and differentiation of the lgl mutant and neighboring wild-type cells, suggesting aberrant cell-cell interactions during development. lgl mutant clones also straddle the anteroposterior boundary of the wing imaginal disc, apparently due to failure of the cells of the anterior and the posterior compartment to segregate at the boundary. To further test if anteroposterior compartmentalization takes place in the neoplastic imaginal discs of lgl mutant larvae, the expression of an engrailed (en)-specific lacZ reporter gene was studied during progressive stages of their tumorous growth. en is activated in the posterior compartments of the neoplastic imaginal discs. However, during later stages of tumorous overgrowth, the en-expressing and nonexpressing cells appear to show extensive intermixing. These observations suggest that neoplastic transformation of imaginal discs involves loss of their normal cell-cell interactions and signaling (Agrawal, 1995).
The lethal(2)giant larvae gene encodes a widely expressed cytoskeletal protein that acts in numerous biological processes during embryogenesis and oogenesis, including cell proliferation, and morphogenetic movements. Having identified the nucleotide change occurring in the l(2)glts3 sequence, the identical change was produced by site-directed mutagenesis leading to the substitution of a serine by a phenylalanine at position 311 of p127l(2)gl and the modified l(2)glF311 gene was introduced into l(2)gl flies. The transgene can fully rescue the development of l(2)gl flies raised at 22°C but causes drastic effects on their development at 29°C confirming the temperature sensitivity of the phenylalanine substitution at position 311. Fertility of females, albeit not of males, is strongly affected. Temperature-shift experiments and microscopic examination of ovaries showed that the mutation blocks egg chamber development at the onset of vitellogenesis (stages 8-9) with growth arrest of the oocyte, incomplete follicle cell migration over the oocyte associated with abnormal organization of the follicular epithelium, and apoptosis of the germline cells, as measured by TUNEL assays. By comparison to wildtype, it was found that p127F311 is already reduced in amount at 22°C and delocalizes from the cytoskeletal matrix, albeit without affecting the apical localization of myosin II, a major partner of p127. At 29°C, the level of p127F311 is even more reduced and the distribution of myosin-II becomes markedly altered at the apices of the follicle cells. These data indicate that during oogenesis p127 plays a critical function at the onset of vitellogenesis and regulates growth of the oocyte, follicle cell migration over the oocyte and their organization in a palisadic epithelium, as well as viability of the germline cells (De Lorenzo, 1999).
During Drosophila metamorphosis, larval tissues, such as the salivary glands, are histolysed whereas imaginal tissues differentiate into adult structures, to form at eclosion a fly-shaped adult. Inactivation of the lethal(2)giant larvae gene encoding the cytoskeletal associated p127 protein, causes malignant transformation of brain neuroblasts and imaginal disc cells with developmental arrest at the larval-pupal transition phase. At this stage, p127 is expressed in wild-type salivary glands that become fully histolysed 12-13 h after pupariation. By contrast to wild-type, administration of 20-hydroxyecdsone to l(2)gl-deficient salivary glands is unable to induce histolysis, although it releases stored glue granules and gives rise to a nearly normal pupariation chromosome puffing, indicating that p127 is required for salivary gland apoptosis. To unravel the l(2)gl function in this tissue, transgenic lines expressing reduced (approximately 0.1) or increased levels of p127 (3.0) were used. The timing of salivary gland histolysis displays an l(2)gl-dose response. Reduced p127 expression delays histolysis whereas overexpression accelerates this process without affecting the duration of third larval instar, prepupal and pupal development. Similar l(2)gl-dependence is noticed in the timing of expression of the cell death genes reaper, head involution defective and grim, supporting the idea that p127 plays a critical role in the implementation of ecdysone-triggered apoptosis. These experiments show also that the timing of salivary gland apoptosis can be manipulated without affecting normal development and provide ways to investigate the nature of the components specifically involved in the apoptotic pathway of the salivary glands (Farkas, 2000).
The abnormal wing discs (awd) gene of Drosophila is homologous to the nm23 gene of mammals, a gene whose expression is altered in metastatic tumors. Both awd and nm23 encode nucleoside diphosphate kinases (NDP kinases). The accumulation of AWD/NDP kinase has been examined during normal development by assaying enzyme activity in extracts. There is a nearly constant level of activity throughout larval and pupal development. The tissue-specific transcription of the awd gene was examined by RNA in situ hybridization and by reporter gene expression. In imaginal discs and brains there is no detectable awd gene expression until the beginning of the third larval instar, despite the constant level of enzyme activity measured in extracts of larvae and pupae. The most intense awd gene expression in imaginal discs and brains occurs after the end of larval development. awd gene expression was examined in neoplastic brain tumors caused by mutations in the lethal (2) giant larvae gene. In l(2)gl mutant brains, as in normal brains, awd gene expression begins during the third larval instar. No tumors form in brains from l(2)gl-;awd- double mutant larva, so awd gene expression is required for tumor formation and/or proliferation. There is more accumulation of AWD/NDP kinase in l(2)gl- mutant brains than there is in normal brains. Using an awd reporter gene, it has been shown that this is a consequence of an increased proportion of awd gene-expressing cells in mutant brains. Using the same awd reporter gene as a marker of donor cells, the invasiveness of l(2)gl-induced neuroblastomas has been confirmed (Timmons, 1993).
Loss of function mutations in the lethal(2)giant larvae gene causes neoplastic brain tumors in Drosophila. A lacZ reporter gene has been introduced into l(2)gl mutant cells and beta-galactosidase expression was used as a marker to monitor the growth of such tumors following transplantation into wild-type adult hosts. Whereas normal larval brains do not grow when transplanted, mutant brains can develop into enormous tumors that fill the entire abdominal cavity. To investigate whether these tumors are similar to mammalian tumors at the biochemical level, the accumulation of a specific protein was examined -- one which is differentially expressed in mammalian metastatic tumors and is likely to be involved in the invasive and/or metastatic mechanism. Increased accumulation of a 72 kilodalton (kDa) type IV collagenase has been observed in several metastatic human tumors. Using antibodies directed against this human 72 kDa type IV collagenase, it has been shown for the first time that Drosophila has a cross-reacting 49 kDa protein with gelatinase activity. In brains dissected from l(2)gl mutant larvae, the accumulation of this 49 kDa gelatinase of Drosophila is increased compared to the level in brains dissected from wild-type larvae. In tumors derived from mutant brains, all of the cells express this protein. Moreover, the tumor cells that invade host organs express this protein. These data suggest that the metastasis of Drosophila tumor cells is similar to the metastasis of some human tumors at the biochemical level as well as at the cellular level (Woodhouse, 1994).
The Drosophila tumor suppressor gene lethal(2) giant larvae (lgl) encodes a cytoskeletal protein required for the change in shape and polarity acquisition of epithelial cells, and also for asymmetric division of neuroblasts. lgl also participates in the release of Decapentaplegic (Dpp), a member of the transforming growth factor ß (TGFß) family that functions in various developmental processes. During embryogenesis, lgl is required for the dpp-dependent transcriptional activation of zipper (zip), which encodes the non-muscle myosin heavy chain (NMHC), in the dorsalmost ectodermal cells -- the leading edge cells. The embryonic expression of known targets of the dpp signaling pathway, such as labial or tinman is abolished or strongly reduced in lgl mutants. lgl mutant cuticles exhibit phenotypes resembling those observed in mutated partners of the dpp signaling pathway. In addition, lgl is required downstream of dpp and upstream of its receptor Thickveins (Tkv) for the dorsoventral patterning of the ectoderm. During larval development, the expression of spalt, a dpp target, is abolished in mutant wing discs, while it is restored by a constitutively activated form of Tkv (TkvQ253D). Taking into account that the activation of dpp expression is unaffected in the mutant, this suggests that lgl function is not required downstream of the Dpp receptor. Finally, the function of lgl responsible for the activation of Spalt expression appears to be required only in the cells that produce Dpp, and lgl mutant somatic clones behave non autonomously. The activity of lgl is therefore positioned in the cells that produce Dpp, and not in those that respond to the Dpp signal. These results are consistent with the same role for lgl in exocytosis and secretion as that proposed for its yeast ortholog, sro7/77 -- lgl might function in parallel or independently of its well-documented role in the control of epithelial cell polarity (Arquier, 2001).
Secretion relies on intracellular vesicular trafficking and on the polarized exocytosis machinery. Recent studies have demonstrated that Lgl function is essential for the establishment of the polarities of epithelial cells. An important issue is therefore to understand whether the role of Lgl in Dpp secretion is direct or simply a consequence of the loss of epithelial cell polarity. Analysis of the temporal requirement for Lgl function argues in favor of Lgl being necessary for the establishment of cell polarity, rather than for its maintenance. Moreover, alteration in Dpp signaling can be observed in lgl mutants in epithelial cells that are correctly polarized and this supports a direct function for Lgl in Dpp secretion (Arquier, 2001).
The epidermis is not affected in homozygous lgl4-null mutant larvae that no longer contain the maternal Lgl protein responsible for a normal embryonic development. lgl4 larvae develop a cuticle that possesses the hallmarks of a wild-type cuticle by all the criteria used, thus indicating that the apical secretion of cuticle components has not been altered. Markers for epithelial cell polarity are localized in the correct position in stage 16 embryos when Lgl is no longer detected. Likewise, lglts3 embryos in which the Lgl protein has lost its cortical location have maintained their typical epithelial cell polarity and their capacity to secrete normal cuticle components. In neuroblasts, Lgl seems to exert its action early during mitosis to recruit basal determinants to the cortex but it does not contribute to their maintenance in this latter location. The polarity of epithelial wing disc cells is preserved until the middle of the third instar larval stage, long after the maternal Lgl contribution has ceased (Arquier, 2001).
It seems reasonable to assume that there is a unique exocytosis pathway mediated by lgl to ensure both cell polarity control and secretion. Dlg and scrib might participate in this same pathway: indeed, they strongly interact genetically with lgl and share with this gene a large panel of identical mutant phenotypes. Lgl, however, does not strictly colocalize with Dlg and Scrib in either epithelial cells. In addition, the Dlg cortical localization does not require lgl function. One could therefore anticipate an lgl action, within a separate and distinct pathway, in parallel to that of dlg and scrib. Further experiments are needed to address this issue (Arquier, 2001).
In yeast, sro7/77-mediated polarized exocytosis relies on a complex regulation and interaction with the actomyosin cytoskeleton. Sro7/77 displays a strong genetic interaction with myo1 (encoding a Type II myosin homolog of NMHC) and with myo2 (encoding an unconventional Type V myosin). In addition, Myo1P can physically interact with Sro7P, in a manner resembling that prevailing between Lgl and Zipper/NMHC. These observations support the idea that Lgl serves as a functional link between the actomyosin cytoskeleton polarity and a specific polarized exocytosis pathway, although the precise function exerted by Lgl in such a process has yet to be deciphered. In yeast, as in flies, myo1 (zipper) and sro7/77 (lgl) display a negative genetic interaction. Loss-of-function alleles of lgl suppress the dorsal closure phenotype in homozygous zip mutants. Conversely, overexpression of lgl enhances the dorsal closure phenotype (Arquier, 2001).
In many developmental processes, polyploid cells are generated by a variation of the normal cell cycle called the endocycle in which cells increase their genomic content without dividing. How the transition from the normal mitotic cycle to endocycle is regulated is poorly understood. The transition from mitotic cycle to endocycle in the Drosophila follicle cell epithelium is regulated by the Notch pathway. Loss of Notch function in follicle cells or its ligand Delta function, in the underlying germline, disrupts the normal transition of the follicle cells from mitotic cycle to endocycle: mitotic cycling continues, leading to overproliferation of these cells. The regulation is at the transcriptional level, since Su(H), a downstream transcription factor in the pathway, is also required cell autonomously in follicle cells for proper transitioning to the endocycle. One target of Notch and Su(H) is likely to be the G2/M cell cycle regulator String, a phosphatase that activates Cdc2 by dephosphorylation. String is normally repressed in the follicle cells just before the endocycle transition, but is expressed when Notch is inactivated. Analysis of the activity of String enhancer elements in follicle cells reveals the presence of an element that promotes expression of String until just before the onset of polyploidy in wild-type follicle cells but well beyond this stage in Notch mutant follicle cells. This suggests that it may be the target of the endocycle promoting activity of the Notch pathway. A second element that is insensitive to Notch regulation promotes String expression earlier in follicle cell development, which explains why Notch, while active at both stages, represses String only at the mitotic cycle-endocycle transition (Deng, 2001).
In Drosophila, mutations in three neoplastic genes, discs large (dlg), lethal(2)giant larvae (lgl) and scribble (scrib), cause loss of apical-basal polarity accompanied by hyperproliferation. It is not understood how loss of apical-basal polarity results in hyperproliferation in these mutants. One attractive hypothesis, however, is that overproliferation is caused by mislocalization of critical plasma membrane proteins that are involved in cell cycle control. The Notch protein is indeed mislocalized throughout lgl mutant cells; this defect could reduce responsiveness to Delta from the germline and thus lead to overproliferation. In addition, many lgl mutant follicle cells existed in multilayered structures where they are less likely to receive the Delta signal because of lack of direct contact with the germline. By contrast, in Notch mutant follicle cells, the cell cycle defect is observed before any obvious polarity defect; normal Armadillo localization is observed in Notch but not in lgl clones. Additional factors probably also contributed to the overproliferation in lgl mutant clones because significantly more proliferation is observed in lgl than in Notch clones (Deng, 2001).
The tumor suppressor genes lethal giant larvae (lgl) and discs large (dlg) act together to maintain the apical basal polarity of epithelial cells in the Drosophila embryo. Neuroblasts that delaminate from the embryonic epithelium require lgl to promote formation of a basal Numb and Prospero crescent, which will be asymmetrically segregated to the basal daughter cell upon division to specify cell fate. Sensory organ precursors (SOPs) also segregate Numb asymmetrically at cell division. Numb functions to inhibit Notch signaling and to specify the fates of progenies of the SOP that constitute the cellular components of the adult sensory organ. In contrast to the embryonic neuroblast, lgl is not required for asymmetric localization of Numb in the dividing SOP. Nevertheless, mosaic analysis reveals that lgl is required for cell fate specification within the SOP lineage; SOPs lacking Lgl fail to specify internal neurons and glia. Epistasis studies suggest that Lgl acts to inhibit Notch signaling by functioning downstream or in parallel with Numb. These findings uncover a previously unknown function of Lgl in the inhibition of Notch and reveal different modes of action by which Lgl can influence cell fate in the neuroblast and SOP lineages (Justice, 2003).
The discovery that lgl function is required to specify cell fate within the SOP lineage, but does not affect asymmetric segregation of Numb, suggests that Lgl function is distinct from Dlg function in the SOP. Lgl function is most likely required after polarization of the SOP and somehow contributes to the selective inhibition of Notch activity that specifies the fate of the pIIb cell. How might Lgl fulfill this function? Lgl is a WD repeat-containing protein conserved in eukaryotes ranging from yeast to man. Similar to many other WD repeat-containing proteins, Lgl likely interacts with multiple partners in a dynamic manner. It binds type II myosins and t-SNAREs on the plasma membrane and is known to be involved in exocytosis in yeast and Drosophila by presumably targeting vesicles to the plasma membrane and thereby inserting membrane proteins at specific zones along the apical-basal axis of epithelial cells and releasing extracellular signaling molecules such as Dpp. The requirement for Lgl function, however, is not restricted to membrane proteins and secreted proteins that require vesicular transport. For example, formation of the basal crescent in neuroblasts involves cytoplasmic and cortical movements of globular proteins, such as Numb, Pon, Prospero, and Miranda, that attach to the cytoplasmic side of the membrane via lipid modifications or association with membrane proteins. One plausible scenario for the role of Lgl in mediating basal Numb crescent formation in neuroblasts is that Lgl and motor proteins form a complex that mediates basal transport of determinants. Such Lgl-containing adaptor complexes in the SOP must differ from those in embryonic neuroblasts under this scenario, given that anterior Numb crescent formation in the SOP is independent of Lgl (Justice, 2003).
A scenario is imagined in which Lgl is required to deliver components of the machinery required for Numb-mediated inhibition of Notch cannot be excluded. Alternatively, Lgl could directly participate in such a mechanism and could perhaps target endocytic vesicles containing Numb and Notch to the lysosome for degradation. A direct role for Lgl in the Notch pathway is supported by studies suggesting that vesicle trafficking of Notch and Delta plays a critical role during Notch pathway signaling. Lgl might bring Notch inhibitors to the plasma membrane or traffic endocytic vesicles in an inhibitory mechanism with Numb and alpha-Adaptin that specifies cell fates in the SOP lineage (Justice, 2003).
Cell fate diversity can be achieved through the asymmetric segregation of cell fate determinants. In the Drosophila embryo, neuroblasts divide asymmetrically and in a stem cell fashion. The determinants Prospero and Numb localize in a basal crescent and are partitioned from neuroblasts to their daughters (GMCs). Nonmuscle myosin II (Zipper) regulates asymmetric cell division by an unexpected mechanism, excluding determinants from the apical cortex. Myosin II is activated by Rho kinase and restricted to the apical cortex by the tumor suppressor Lethal (2) giant larvae. During prophase and metaphase, myosin II prevents determinants from localizing apically. At anaphase and telophase, myosin II moves to the cleavage furrow and appears to ìpushî rather than carry determinants into the GMC. Therefore, the movement of myosin II to the contractile ring not only initiates cytokinesis but also completes the partitioning of cell fate determinants from the neuroblast to its daughter (Barros, 2003).
Class II myosins are barbed end-directed motors that form bipolar filaments. The filaments bind actin and initiate contraction when the two ends of the bipolar filament pull in opposite directions. Myosin's mode of action makes it unlikely that myosin II could transport cargo from one side of the cell to the other, except perhaps by progressive contraction along the cortex. The lack of colocalization of myosin II and Miranda in neuroblasts further implies that myosin II does not transport Miranda directly. The data suggest, first, that myosin II is required to maintain an intact cortical actin cytoskeleton and, second, that active myosin modifies the actin cytoskeleton at the apical cortex to exclude Miranda binding. The C. elegans myosin II may act in a similar fashion, as it appears to limit PAR-3 to the anterior of the zygote (Barros, 2003).
Several alternative approaches were taken to inactivate myosin II in neuroblasts. First, germline clones of Sqh were analyzed. In severe sqh1GLC embryos, levels of the regulatory light chain are greatly reduced from early development, and the heavy chain is found only in inactive aggregates. The actin cytoskeleton is disrupted, and neither Lgl nor Miranda localize to the cortex. Miranda concentrates instead at the spindle microtubules. Therefore, active myosin is necessary from early development to organize the actin cytoskeleton, which is in turn required for Lgl and Miranda to localize to the cortex (Barros, 2003).
Myosin II is activated by phosphorylation of its regulatory light chain by Rho kinase. Myosin was inactivated at the time of neuroblast cell division by inhibition of Rho kinase. Myosin II no longer localizes at the apical neuroblast cortex but instead spreads into the cytoplasm, and basal protein localization is disrupted. Although F-actin and Lgl remain uniformly at the cortex, cell fate determinants are now found around the entire cell cortex, demonstrating that apical cortical myosin is required to confine determinants to the basal half of the cell. Inhibition of Rho kinase also blocks cytokinesis, although the defect in basal protein localization is unlikely to be the consequence of mitotic arrest or a block in cytokinesis. First, basal protein localization is not disrupted in neuroblasts arrested in mitosis by colcemid treatment. Second, mitosis occurs without cytokinesis in pebble mutants, but the resultant polyploid neuroblasts still localize Numb and Prospero asymmetrically. Finally, the loss of asymmetry resulting from Rho kinase inhibition can be rescued by expression of a constitutively active form of the myosin II regulatory light chain (SqhE20E21). It is concluded that myosin II is required to restrict cell fate determinants to the basal cortex (Barros, 2003).
Myosin II localizes to the apical cortex of metaphase neuroblasts. Why is myosin localization/activity asymmetric? Lgl binds myosin II heavy chain directly and inhibits myosin filament formation. This binding is regulated by phosphorylation of Lgl; this phosphorylation inhibits its interaction with myosin II in vitro. If Lgl negatively regulates myosin activity and localization, then myosin should be uniformly distributed in an lgl mutant. Indeed, in lgl1GLC mutants myosin II no longer concentrates apically but is found uniformly around the cortex. Most Miranda protein is released from the cortex and binds microtubules, again suggesting that myosin excludes Miranda from the cortex (Barros, 2003).
Myosin II localizes to the entire cortex in lgl mutants and thereby prevents Miranda binding basally. In Drosophila neuroblasts in which Lgl levels are reduced (zygotic lgl1 mutants), Miranda is released from the cortex. Miranda localization can be rescued by simultaneously reducing the level of myosin II (zip1 zygotic mutants). Reducing the level of active myosin may restore the balance between the levels of Lgl and myosin, enabling the remaining myosin to concentrate apically (Barros, 2003).
How does myosin II restrict neuroblast proteins to the basal side of the cell cortex? Myosin II and Miranda occupy primarily opposite sides at the neuroblast cortex: myosin II is concentrated at the apical cortex while Miranda localizes as a basal crescent. As myosin II shifts to the cleavage furrow, Miranda is segregated into the forming GMC. The apical F-actin compartment may be modified by myosin II to exclude binding of basal proteins like Miranda. Active myosin II requires Rho kinase activity and depends on inactivation of Lgl at the apical cortex by aPKC (Betschinger, 2003). Ectopic expression of a nonphosphorylatable form of Lgl, in which the conserved aPKC-dependent phosphorylation sites are mutated from Serines to Alanines (Lgl-3A), results in mislocalization of Miranda around the neuroblast cortex (Betschinger, 2003). The data support a spatially regulated interaction between myosin II and Lgl. Myosin is apically localized in wild-type neuroblasts, corresponding to the domain in which Lgl is inactivated by aPKC. In lgl mutants, myosin is no longer restricted apically but localizes around the entire cell cortex. Conversely, when nonphosphorylatable Lgl is expressed in neuroblasts, myosin is inhibited throughout the cell and drops off the cortex. It is proposed that myosin II is activated and can form filaments at the apical cortex, where phosphorylated Lgl is inactive and unable to bind myosin II. Myosin may then modify the actin cytoskeleton to prevent the binding of Miranda. At the basal cortex, in the absence of aPKC, Lgl is active and can bind and inhibit myosin. Myosin cannot form filaments, which are required for it to bind to the actin cortex. As a result, Miranda can bind to the basal cortex (Barros, 2003).
At anaphase, myosin II moves to the equator and appears to “push” cell fate determinants into the daughter cell. This movement is regulated in an Lgl-independent fashion and occurs whether myosin is restricted to the apical cortex or is uniformly cortical (as in lgl mutants). Cortical myosin is essential, however, to efficiently segregate determinants into the GMC at telophase (telophase rescue). In neuroblasts expressing Lgl-3A, myosin II is cytoplasmic, and determinants are not partitioned to the daughter cell. Nonetheless, at telophase, myosin seems to be recruited from the cytoplasm, since it still accumulates to the cleavage furrow. Thus three separate steps of myosin regulation in neuroblasts can be defined. First, myosin forms an apical crescent. This is positively regulated by Rho kinase and negatively regulated by Lgl. Second, cortical myosin moves to the equator. This movement occurs independently of Lgl. Third, cortical and cytoplasmic myosin accumulates at the cleavage furrow, a step that is also Lgl independent. Rho Kinase activation seems to be important for all three steps of myosin II regulation. When Rho kinase is inhibited, myosin falls into the cytoplasm, and there is no cleavage furrow formation (Barros, 2003).
In conclusion, these results demonstrate that myosin II acts downstream of Lgl and the apical protein complex to regulate the segregation of cell fate determinants. Myosin II does not negatively regulate basal protein targeting, as has previously been suggested nor does it transport determinants directly. Instead, it is proposed that myosin II acts in a novel fashion, excluding determinants from the apical cortex and 'pushing' them into the GMC at anaphase and telophase. Myosin II might modify the actin cytoskeleton to prevent determinants binding, although the actual structure formed and the physical change in the actin cytoskeleton remains to be determined (Barros, 2003).
Cell polarity is essential for generating cell diversity and for the proper function of most differentiated cell types. In many organisms, cell polarity is regulated by the atypical protein kinase C (aPKC), Bazooka (Baz/Par3), and Par6 proteins. Drosophila aPKC zygotic null mutants survive to mid-larval stages, where they exhibit defects in neuroblast and epithelial cell polarity. Mutant neuroblasts lack apical localization of Par6 and Lgl, and fail to exclude Miranda from the apical cortex; yet, they show normal apical crescents of Baz/Par3, Pins, Inscuteable, and Discs large and normal spindle orientation. Mutant imaginal disc epithelia have defects in apical/basal cell polarity and tissue morphology. In addition, aPKC mutants show reduced cell proliferation in both neuroblasts and epithelia, the opposite of the lethal giant larvae (lgl) tumor suppressor phenotype; reduced aPKC levels strongly suppress most lgl cell polarity and overproliferation phenotypes (Rolls, 2003).
One of the more unexpected findings is that aPKC mutant neuroblasts show normal Baz/Par3 apical localization. The Baz/Par3-Par6-aPKC complex has been suggested to form a functional unit that is interdependent for localization in C. elegans, mammals, and Drosophila. Baz/Par3 shows normal apical localization in aPKC mutant neuroblasts, showing that normal Baz/Par3 localization can occur without being part of the Par3-Par6-aPKC complex. In addition, neuroblasts lacking apical aPKC and Par6 still form a molecularly defined apical cortical domain containing Baz/Par3, Insc, Pins, and Dlg. These results lead to the proposal of a hierarchy for apical protein localization in neuroblasts: Baz/Par3-Insc-Pins-Dlg --> aPKC --> Par6-Lgl. This hierarchy is consistent with recent biochemical analyses in which a protein complex was isolated containing Par6-aPKC-Lgl, but not Baz/Par3. It is suggested that aPKC may be required to anchor the Par6-aPKC-Lgl complex at the apical cortex of the neuroblast (Rolls, 2003).
Both aPKC and Lgl are required for Miranda basal localization in neuroblasts, and all available data support a model in which Lgl is required for targeting Miranda to the neuroblast cortex, whereas aPKC blocks Lgl function on the apical side of the neuroblast: (1) lgl mutants have little or no Miranda at the cortex; (2) aPKC mutants show uniform cortical Miranda localization; (3) a weak lgl phenotype can be suppressed by reducing aPKC levels, showing that aPKC activity antagonizes Lgl activity; (4) aPKC and Lgl physically interact; (5) an overexpressed nonphosphorylatable Lgl protein is uniformly cortical and able to induce uniform cortical Miranda localization, whereas phospho-Lgl is preferentially released from the cell cortex. This has led to a model in which Lgl acts as an anchor for Miranda at the basal cortex, but is absent from the apical cortex due to aPKC-mediated phosphorylation. Although this simple model is attractive, it is noted that Lgl has never been observed colocalized with Miranda in a basal cortical crescent, and a role for cytoplasmic Lgl in Miranda localization has not been definitively ruled out (Rolls, 2003).
The Baz/Par3-Par6-aPKC complex has a well-characterized role in regulating neuroblast spindle orientation. Spindle orientation can be measured relative to extrinsic landmarks around the neuroblast (e.g., perpendicular to the overlying ectoderm) or relative to intrinsic cues within each neuroblast (e.g., perpendicular to the Baz/Par3-Par6-aPKC apical crescent). Mutations in baz or par6 genes randomize embryonic neuroblast spindle orientation relative to the overlying ectoderm, but it is not clear whether these phenotypes are due to disruption of the ectodermal layer or to a cell-autonomous defect in the neuroblast. The function of aPKC in embryonic neuroblast spindle orientation cannot be assayed due to high levels of maternal aPKC protein present in aPKC zygotic mutant embryos. However, aPKC is not required for intrinsic spindle orientation in larval neuroblasts; the mitotic spindle is always perpendicular to the Baz/Par3-Insc-Pins apical crescent (Rolls, 2003).
The Baz/Par3 and Par6 proteins are required to establish epithelial polarity in Drosophila, and aPKC is also shown to be required for normal apical/basal epithelial cell polarity. The similar phenotype in baz, par6, and aPKC mutants may indicate that these proteins function together as a complex to provide a single function in epithelia, despite the evidence that they have independent functions in neuroblasts. One primary function may be the inhibition of Lgl activity because it is found that the lgl epithelial polarity defects can be strongly suppressed by reducing aPKC levels. Lgl-Dlg-Scrib activity can also antagonize Baz/Par3-Par6-aPKC activity, and it is tempting to speculate that aPKC inactivates Lgl by phosphorylation, whereas Lgl can inactivate aPKC by sequestering it into an Lgl-Par6-aPKC complex and out of the Baz/Par3-Par6-aPKC complex (Rolls, 2003).
The role of aPKC in cell proliferation has not been previously investigated in Drosophila. There are three lines of evidence showing that aPKC promotes cell proliferation in neuroblasts and epithelia. The number of cells in aPKC mutant mushroom body neuroblast clones is significantly lower than the number in wild-type clones. There appears to be a normal number of early-born gamma neurons in these clones, followed by only a few later-born alpha neurons. The normal number of early-born gamma neurons suggests that loss of aPKC does not lead to cell death in this population; moreover, no decrease is seen in the number of neuroblasts per brain lobe in aPKC mutant larvae. It is concluded that cell death is not contributing to the reduction in neurons observed in the clones, but rather, that the neuroblast stops dividing near the time the neuroblast switches over to generating alpha and ß neurons. The neuroblast may become arrested at some point in the cell cycle, or it may undergo a terminal division to generate a pair of GMCs (perhaps due to both daughter cells inheriting Miranda and Prospero GMC determinants). A second indication that aPKC promotes cell proliferation is that far fewer epithelial cells are observed in aPKC mutant eye imaginal discs compared with the wild type, even with an additional day of growth as second instar larvae. Finally, a 50% reduction in aPKC levels (aPKC/+) can strongly suppress the epithelial and brain overproliferation phenotypes of lgl mutants. Together, these data show that aPKC positively regulates cell proliferation in epithelia and neuroblasts. Interestingly, reduction in the function of the mammalian atypical PKCzeta (using overexpression of a dominant-negative kinase) can suppress Rac1/cdc42-induced overproliferation. Thus, aPKC may have an evolutionarily conserved role in promoting cell proliferation, as well as in the establishment of cell polarity (Rolls, 2003).
Although aPKC and Lgl act antagonistically to regulate many aspects of epithelial and neuroblast cell polarity and cell proliferation, they may share a common positive function in regulating neuroblast apical cell size. lgl zygotic mutants have some embryonic telophase neuroblasts with an abnormally small apical cortical domain, apical spindle pole, and neuroblast size. These defects are not suppressed by reducing aPKC levels, and in fact may be enhanced. Thus, it is proposed that Lgl and aPKC both act positively to promote large apical cell and spindle pole size. It has been reported that Baz/Par3-Par6-aPKC and Pins-Gαi act in parallel pathways to promote large apical cell size and apical spindle size; Lg could be acting as part of the Pins-Galphai pathway, or as a third input promoting apical cell and spindle size (Rolls, 2003).
How a cell chooses to proliferate or to differentiate is an important issue in
stem cell and cancer biology. Drosophila neuroblasts undergo self-renewal
with every cell division, producing another neuroblast and a differentiating
daughter cell, but the mechanisms controlling the self-renewal/differentiation
decision are poorly understood. This study tested whether cell polarity genes,
known to regulate embryonic neuroblast asymmetric cell division,
also regulate neuroblast self-renewal. Clonal analysis
in larval brains shows that pins mutant neuroblasts rapidly fail to
self-renew, whereas lethal giant larvae (lgl) mutant neuroblasts
generate multiple neuroblasts. Notably, lgl pins double mutant
neuroblasts all divide symmetrically to self-renew, filling the brain with
neuroblasts at the expense of neurons. The lgl pins neuroblasts show
ectopic cortical localization of atypical protein kinase C (aPKC), and a
decrease in aPKC expression reduces neuroblast numbers, suggesting that aPKC
promotes neuroblast self-renewal. In support of this hypothesis,
neuroblast-specific overexpression of membrane-targeted aPKC, but not a
kinase-dead version, induces ectopic neuroblast self-renewal. It is concluded that
cortical aPKC kinase activity is a potent inducer of neuroblast
self-renewal (Lee, 2005).
Drosophila neuroblasts are an
excellent model system in which to investigate the molecular control of
self-renewal versus differentiation. Larval neuroblasts repeatedly divide
asymmetrically to self-renew a neuroblast and to produce a smaller daughter
cell, called a ganglion mother cell (GMC), that typically makes two postmitotic
neurons; this process enables a single neuroblast to generate many hundreds of
neurons. Self-renewal is defined as the capacity of a
neuroblast to maintain all attributes of its cell type (molecular markers and
proliferation potential). In this regard, a neuroblast is very similar to a
germline stem cell: both maintain their stem cell identity while generating
differentiating progeny. About 100 neuroblasts per
brain lobe are formed during embryogenesis, where they proliferate briefly
before entering quiescence. Brain neuroblasts
re-enter the cell cycle between 10 and 72 h after larval hatching
(ALH), and then a stable population of ~100 mitotic, self-renewing neuroblasts is
maintained. This invariant neuroblast
number was used to screen for mutants altering self-renewal versus differentiation:
mutants in which a neuroblast makes two neuroblast progeny (ectopic
self-renewal) will have >100 neuroblasts, whereas mutants in which a
neuroblast makes two GMC progeny (failure in self-renewal) will have <100
neuroblasts. This assay was used to test known cell polarity mutants
for a role in neuroblast self-renewal (Lee, 2005).
Two classes of cell
polarity regulators were assayed for an effect on larval neuroblast self-renewal.
lgl and discs large (dlg) zygotic mutants were examined, because
these mutants form brain tumours and promote basal
protein targeting in embryonic and larval neuroblasts. Lgl
and Dlg have several protein interaction motifs and are localized around the
neuroblast cortex. In addition, pins
and Galphai zygotic mutants were examined; these genes regulate cell
polarity in embryonic neuroblasts, but have not
been well characterized in larval neuroblasts. Pins and Galphai are
colocalized with Inscuteable and the evolutionarily conserved
Bazooka-Par6- aPKC proteins at the apical cortex of mitotic
neuroblasts, and all of these proteins are partitioned into the neuroblast
during cytokinesis (Lee, 2005).
In wild-type larvae, a population of ~100 neuroblasts
could be identified by the markers Worniu,
Deadpan and Miranda, and by labelling with a pulse of 5-bromodeoxyuridine
(BrdU); by contrast, the thousands of differentiating GMCs and neurons rapidly
downregulate neuroblast markers and express nuclear Prospero and/or Elav.
A clear increase in neuroblast number is observed in
lgl and dlg mutants; there are supernumerary neuroblasts at all
stages examined; all extra neuroblasts expressed Deadpan and Miranda and
are proliferative on the basis of their ability to incorporate BrdU. Galphai
zygotic mutants have a complex phenotype that will be described
in a later publication; however, pins zygotic mutants show a marked decrease in
neuroblast number. Notably, this phenotype is not due to a subset of
neuroblasts remaining quiescent, because neuroblast numbers peak and then
decline over time, and it is not due to neuroblast
cell death. The relatively late onset of the pins phenotype
is probably due to the gradual depletion of maternal pins gene product
in these larvae (Lee, 2005).
To determine whether the pins and lgl larval brain phenotypes are
due to defects in neuroblast self-renewal, positively marked genetic
clones were induced in single neuroblasts to trace their
progeny. Clone induction
parameters were adjusted to ensure that each clone was derived from a single neuroblast (1.2
clones per lobe). In wild-type brains, neuroblast clones always
contained a single Worniu+ Miranda+
nuclear-Prospero- neuroblast and numerous smaller Worniu-
Miranda- nuclear-Prospero+ progeny,
confirming that wild-type neuroblasts always
divide to self-renew and to generate a smaller differentiating GMC.
By contrast, lgl mutant brains had an average of
2.3 neuroblasts per clone, with up to six neuroblasts per clone, showing that
lgl mutant neuroblasts can divide symmetrically to yield two neuroblasts.
The opposite phenotype was seen in pins
mutant brains: 72.8% of the clones had no neuroblast and the remainder had a
single neuroblast.
The neuroblasts did not die in the pins mutants as evidenced by the following: the cell death marker
caspase-3 was not upregulated, neuroblast-specific expression of the p35 cell death inhibitor
did not rescue the missing neuroblasts, and one
clone was observed in which the largest cell coexpressed neuroblast and GMC markers,
consistent with an intermediate stage in neuroblast-to-GMC differentiation. It is concluded
that wild-type neuroblasts exclusively
generate neuroblast/GMC siblings; lgl mutant neuroblasts occasionally
undergo ectopic self-renewal to generate neuroblast/neuroblast siblings; and
pins mutant neuroblasts occasionally fail to self-renew, resulting in
GMC/GMC siblings and termination of the lineage (Lee, 2005).
Next to be examined was whether lgl pins double mutants had fewer neuroblasts (like
pins mutants) or extra neuroblasts (like lgl mutants).
Unexpectedly, a phenotype was detected in which the larval brain was full of
cells expressing the neuroblast markers Worniu, Miranda and Deadpan and lacking
expression of the neuronal marker Elav. Additional markers that distinguish neuroblasts and
GMCs were examined to determine whether these cells were neuroblasts or a hybrid
neuroblast/GMC identity. Both wild-type neuroblasts and lgl
pins cells actively transcribed the worniu, deadpan,
miranda and prospero genes, maintained proliferation, did not
express the Elav neuronal differentiation marker, and did not extend axons.
The only potential GMC attribute found in lgl pins
neuroblasts was nuclear Prospero protein but, because wild-type
neuroblasts and GMCs both contain Prospero protein, which can accumulate in
neuroblast nuclei if not properly localized, this protein is not a definitive
marker for the GMC cell type. Thus, lgl pins brains contain large numbers
of ectopic, proliferating, self-renewing neuroblasts. Combining these
lgl, pins and lgl pins mutant data leads to the conclusion
that Lgl inhibits self-renewal, whereas Pins has dual functions in promoting and
inhibiting self-renewal (Lee, 2005).
To understand how Lgl and Pins
regulate neuroblast self-renewal at the cellular level, cortical
polarity marker localization was examined in mitotic larval neuroblasts. In wild-type larval
neuroblasts, the Par complex (Bazooka-Par6-aPKC) and
Pins-Galphai proteins forms an apical crescent at metaphase
and are partitioned into the self-renewing neuroblast at telophase, whereas the
Miranda and Prospero proteins form a basal crescent at metaphase and are
partitioned into the differentiating GMC at telophase. In lgl
pins double mutants, in which all neuroblasts divide symmetrically to
generate self-renewing neuroblast/neuroblast siblings, most mitotic neuroblasts
show uniform cortical aPKC, cytoplasmic Bazooka and Par6, and uniform cortical
Miranda at metaphase and telophase.
Thus, only aPKC maintained its correct subcellular localization and correlated
with neuroblast self-renewal (Lee, 2005).
aPKC localization was examined in lgl and pins single mutants, in which symmetric
divisions occurred at lower frequency. In lgl mutants, aPKC showed weak
ectopic cortical localization in about half the metaphase neuroblasts, whereas
Miranda was delocalized from the cortex; by telophase, however, both proteins
appeared to be localized normally. Ectopic cortical
aPKC was also observed in dlg mutant larval neuroblasts.
A role for Lgl in restricting aPKC to the apical cortex of neuroblasts has not
been reported but
would be consistent with the observation that basolateral Lgl restricts aPKC to
the apical surface of Drosophila and vertebrate epithelia and
Xenopus blastomeres. In pins mutants, aPKC and cytoplasmic Miranda
showed weak uniform cortical distribution in metaphase neuroblasts, but were properly
localized in most telophase neuroblasts
Thus, both Lgl and Pins are required to restrict aPKC to the apical cortex in
metaphase neuroblasts (Lee, 2005).
Whether aPKC is required for neuroblast self-renewal was examined.
aPKC mutant clones in larval
mushroom body neuroblasts showed premature lineage termination,
consistent with aPKC being required for neuroblast
self-renewal. In addition, aPKC null mutants died as second instar larvae
with reduced neuroblast numbers. Because this was a
relatively mild phenotype and there was no detectable aPKC protein at this
stage, it is likely that there are additional pathways for stimulating
neuroblast self-renewal. Next, whether aPKC is required for ectopic
neuroblast self-renewal in the lgl mutants was tested. lgl aPKC double
mutants had normal numbers of neuroblasts, showing
that aPKC is required for the ectopic neuroblast self-renewal seen in lgl
mutants. aPKC mutants also suppressed ectopic neuroblast self-renewal in
several independently isolated lgl mutations, further supporting a role
for aPKC in self-renewal. In addition, it was found that aPKC is fully epistatic to
lgl in regulating Miranda localization. Thus, aPKC is required for the ectopic
neuroblast self-renewal and Miranda delocalization phenotypes seen in lgl
mutants (Lee, 2005).
These data are most consistent with a model in which
Lgl negatively regulates aPKC, and aPKC directly promotes self-renewal. This
model is based on the observations that Lgl restricts aPKC localization to the
apical cortex of neuroblasts and that a reduction in aPKC blocks the lgl
self-renewal phenotype. To test this model,
worniu-Gal4 line was used to drive neuroblast-specific expression of
constitutively active aPKC or Lgl proteins, and an increase or
decrease in neuroblast numbers was assayed. Neuroblast-specific expression of aPKC targeted
to the plasma membrane with a CAAX prenylation motif
(UAS-aPKCCAAXWT)
resulted in ectopic cortical aPKC localization, loss of
cortical Miranda, and a large increase in the number
of neuroblasts. These effects were not observed after overexpression of wild-type
aPKC or a membrane-targeted kinase-dead aPKC
(UAS-aPKCCAAXKD).
Expression of a constitutively active aPKC
(UAS-aPKCDeltaN) that was predominantly
cytoplasmic gave only a slight increase in
neuroblast number, showing that cortical
localization of aPKC is essential to generate ectopic neuroblasts. By contrast,
neuroblast-specific expression of a constitutively active Lgl protein (Lgl3A)
resulted in the expected uniform cortical localization of Miranda, but no change in
neuroblast numbers. Combined overexpression of both
Lgl3A and aPKCCAAXWT, however, resulted in strong suppression of the
aPKCCAAXWT ectopic neuroblast phenotype,
even though Lgl3A alone had no effect on neuroblast
numbers, consistent with Lgl inhibiting aPKC function either directly or
through its downstream effectors. Thus, neuroblast-specific overexpression of
aPKC can expand the neuroblast population (most probably by promoting symmetric
neuroblast/neuroblast cell divisions) without eliminating the ability of these
neuroblasts to undergo asymmetric neuroblast/GMC divisions to generate
differentiating progeny. It is concluded that aPKC is sufficient to promote
neuroblast self-renewal, Lgl can inhibit aPKC function, and membrane-targeting
and kinase activity are essential for aPKC function (Lee, 2005).
This study has established
Drosophila larval neuroblasts as a model system for studying self-renewal
versus differentiation. A simple model is proposed in which Pins anchors
aPKC apically and Lgl inhibits aPKC localization basally, thereby restricting
aPKC to the apical cortex where it promotes neuroblast self-renewal.
In addition, aPKC can phosphorylate and directly
inhibit Lgl function, which together with the current
data provides evidence for mutual inhibition between Lgl and aPKC in
neuroblasts, similar to the mutual inhibition seen between these two proteins in
epithelia. Mutual inhibition between aPKC and Lgl would result in stabilization of apical
aPKC localization and more reliable partitioning of aPKC into the neuroblast
during mitosis. In pins mutants, aPKC is delocalized and nonfunctional
owing to Lgl activity, thereby reducing self-renewal; in lgl mutants,
aPKC shows weak ectopic cortical localization that increases self-renewal, and
in lgl pins double mutants, aPKC is both delocalized and fully active:
thus all neuroblasts undergo symmetric self-renewal.
Although the targets of aPKC involved in self-renewal are unknown, aPKC
may directly phosphorylate and inactivate GMC determinants,
and/or phosphorylate and activate neuroblast-specific
proteins. Notably, lgl1 mutant mice have neural progenitor
hypertrophy and knockdown of a pins
mammalian homologue (AGS3) leads to depletion of neural progenitors:
phenotypes that are very similar to those described
in this study. In the future, it will be important to determine the role of aPKC in
mammalian neural progenitor self-renewal and to identify the aPKC-regulated
phosphoproteins that regulate neuroblast self-renewal in
Drosophila (Lee, 2005).
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date revised: 17 January 2008
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