Cell behaviour of Drosophila fat cadherin mutations in wing development

We have studied several cell behaviour parameters of mutant alleles of fat (ft) in Drosophila imaginal wing disc development. Mutant imaginal discs continue growing in larvae delayed in pupariation and can reach sizes of several times those of wild-type. Their growth is, however, basically allometric. Homozygous ft cells grow faster than their twin cells in clones and generate larger territories, albeit delimited by normal clonal restrictions. Moreover, ft cells in clones tend to grow towards wing proximal regions. These behaviours can be related with failures in cell adhesiveness and cell recognition. Double mutant combinations with alleles of other genes, e.g. of the Epidermal growth factor receptor (DER) pathway, modify ft clonal phenotypes, indicating that adhesiveness is modulated by intercellular signalling. Mutant ft cells show, in addition, smaller cell sizes during proliferation and abnormal cuticular differentiation, which reflect cell membrane and cytoskeleton anomalies, which are not modulated by the DER pathway.     

Transvection in the Drosophila Ultrabithorax Gene: A Cbx(1) Mutant Allele Induces Ectopic Expression of a Normal Allele in Trans

In wild-type Drosophila melanogaster larvae, the Ultrabithorax (Ubx) gene is expressed in the haltere imaginal discs but not in the majority of cells of the wing imaginal discs. Ectopic expression of the Ubx gene in wing discs can be elicited by the presence of Contrabithorax (Cbx) gain-of-function alleles of the Ubx gene or by loss-of-function mutations in Polycomb (Pc) or in other trans-regulatory genes which behave as repressors of Ubx gene activity. Several Ubx loss-of-function alleles cause the absence of detectable Ubx proteins (UBX) or the presence of truncated UBX lacking the homeodomain. We have compared adult wing phenotypes with larval wing disc UBX patterns in genotypes involving double mutant chromosomes carrying in cis one of those Ubx mutations and the Cbx1 mutation. We show that such double mutant genes are (1) active in the same cells in which the single mutant Cbx1 is expressed, although they are unable to yield functional proteins, and (2) able to induce ectopic expression of a normal homologous Ubx allele in a part of the cells in which the single mutant Cbx1 is active. That induction is conditional upon pairing of the homologous chromosomes (the phenomenon known as transvection), and it is not mediated by UBX. Depletion of Pc gene products by Pc3 mutation strongly enhances the induction phenomenon, as shown by (1) the increase of the number of wing disc cells in which induction of the homologous allele is detectable, and (2) the induction of not only a paired normal allele but also an unpaired one.     

Roles for scalloped and vestigial in regulating cell affinity and interactions between the wing blade and the wing hinge

The scalloped and vestigial genes are both required for the formation of the Drosophila wing, and recent studies have indicated that they can function as a heterodimeric complex to regulate the expression of downstream target genes. We have analyzed the consequences of complete loss of scalloped function, ectopic expression of scalloped, and ectopic expression of vestigial on the development of the Drosophila wing imaginal disc. Clones of cells mutant for a strong allele of scalloped fail to proliferate within the wing pouch, but grow normally in the wing hinge and notum. Cells overexpressing scalloped fail to proliferate in both notal and wing-blade regions of the disc, and this overexpression induces apoptotic cell death. Clones of cells overexpressing vestigial grow smaller or larger than control clones, depending upon their distance from the dorsal-ventral compartment boundary. These studies highlight the importance of correct scalloped and vestigial expression levels to normal wing development. Our studies of vestigial-overexpressing clones also reveal two further aspects of wing development. First, in the hinge region vestigial exerts both a local inhibition and a long-range induction of wingless expression. These and other observations imply that vestigial-expressing cells in the wing blade organize the development of surrounding wing-hinge cells. Second, clones of cells overexpressing vestigial exhibit altered cell affinities. Our analysis of these clones, together with studies of scalloped mutant clones, implies that scalloped- and vestigial-dependent cell adhesion contributes to separation of the wing blade from the wing hinge and to a gradient of cell affinities along the dorsal-ventral axis of the wing.     

Mutations in Genes Encoding Essential Mitotic Functions in DROSOPHILA MELANOGASTER

Temperature-sensitive mutations at 15 loci that affect the fidelity of mitotic chromosome behavior have been isolated in Drosophila melanogaster. These mitotic mutants were detected in a collection of 168 EMS-induced X-linked temperature-sensitive (ts) lethal and semilethal mutants. Our screen for mutations with mitotic effects was based upon the reasoning that under semirestrictive conditions such mutations could cause an elevated frequency of mitotic chromosome misbehavior and that such events would be detectable with somatic cell genetic techniques. Males hemizygous for each ts lethal and heterozygous for the recessive autosomal cell marker mwh were reared under semirestrictive conditions, and the wings of those individuals surviving to adulthood were examined for an increased frequency of mwh clones. Those mutations producing elevated levels of chromosome instability during growth of the wing imaginal disc were also examined for their effects on chromosome behavior in the cell lineages producing the abdominal cuticle. Fifteen mutations affect chromosome behavior in both wing and abdominal cells and thus identify loci generally required for the fidelity of mitotic chromosome transmission. Mapping and complementation tests show that these mutations represent 15 loci. One mutant is an allele of a locus (mus-101) previously identified by mutagen-sensitive mutants and a second mutant is an allele of the lethal locus zw 10.--The 15 mutants were also examined cytologically for their effects on chromosomes in larval neuroblasts. Taken together, the results of our cytological and genetical studies show that these mutants identify loci with wild-type functions necessary for either maintenance of chromosome integrity or regular disjunction of chromosomes or chromosome condensation. Thus, these mutations define a broad spectrum of genes required for the normal execution of the mitotic chromosome cycle.     

The Drosophila segment polarity gene patched interacts with decapentaplegic in wing development.

The decapentaplegic (dpp) gene of Drosophila melanogaster encodes a polypeptide of the transforming growth factor-beta family of secreted factors. It is required for the proper development of both embryonic and adult structures, and may act as a morphogen in the embryo. In wing imaginal discs, dpp is expressed and required in a stripe of cells near the anterior-posterior compartment boundary. Here we show that viable mutations in the segment polarity genes patched (ptc) and costal-2 (cos2) cause specific alterations in dpp expression within the anterior compartment of the wing imaginal disc. The interaction between ptc and dpp is particularly interesting; both genes are expressed with similar patterns at the anterior-posterior compartment boundary of the disc, and mis-expressed in a similar way in segment polarity mutant backgrounds like ptc and cos2. This mis-expression of dpp could be correlated with some of the features of the adult mutant phenotypes. We propose that ptc controls dpp expression in the imaginal discs, and that the restricted expression of dpp near the anterior-posterior compartment boundary is essential to maintain the wild-type morphology of the wing disc.     

Effect of mutations in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> tumor suppressor <Emphasis Type="Italic">Merlin</Emphasis> on proliferation and differentiation of wing cells

Experiments on transplantation of wing imaginal discs homozygous for a mutation in the tumor suppressor gene Merlin have demonstrated that this mutation does not induce malignant tumors. Marking of the wing disc compartment borders by specific antibodies showed the absence of essential compartment border defects in case of the Merlin mutation. Drosophila melanogaster cells mutant for Merlin have shorter cell cycle than normal cells. Proliferation of imaginal discs lasts longer in case of the mutation. It is known that beginning from some moment of development, wing veins serve as clonal restriction lines that cannot be crossed by growing mosaic clones. We showed that the Merlin mutation leads to depression of vein clonal restriction property. This means that this gene is involved not only in the control of cell proliferation, but also in the control of cell mobility and adhesion.     

Screening of larval/pupal P-element induced lethals on the second chromosome in Drosophila melanogaster : clonal analysis and morphology of imaginal discs

We have carried out screens for lethal mutations on the second chromosome of Drosophila melanogaster that are associated with abnormal imaginal disc morphologies, particularly in the wing disc. From a collection of 164 P element-induced mutations with a late larva/pupa lethal phase we have identified 56 new loci whose gene products are required for normal wing disc development and for normal morphology of other larval organs. Genetic mosaics of these 56 mutant lines show clonal mutant phenotypes for 23 cell-viable mutations. These phenotypes result from altered cell parameters. Causal relationships between disc and clonal phenotypes are discussed. Received: 19 June 1997 / Accepted: 4 August 1997     

Mutations That Alter the Timing and Pattern of Cubitus Interruptus Gene Expression in Drosophila Melanogaster

The cubitus interruptus (ci) gene is a member of the Drosophila segment polarity gene family and encodes a protein with a zinc finger domain homologous to the vertebrate Gli genes and the nematode tra-1 gene. Three classes of existing mutations in the ci locus alter the regulation of ci expression and can be used to examine ci function during development. The first class of ci mutations causes interruptions in wing veins four and five due to inappropriate expression of the ci product in the posterior compartment of imaginal discs. The second class of mutations eliminates ci protein early in embryogenesis and causes the deletion of structures that are derived from the region including and adjacent to the engrailed expressing cells. The third class of mutations eliminates ci protein later in embryogenesis and blocks the formation of the ventral naked cuticle. The loss of ci expression at these two different stages in embryonic development correlates with the subsequent elimination of wingless expression. Adults heterozygous for the unique ci(Ce) mutation have deletions between wing veins three and four. A similar wing defect is present in animals mutant for the segment polarity gene fused that encodes a putative serine/threonine kinase. In ci(Ce)/+ and fused mutants, the deletions between wing veins three and four correlate with increased ci protein levels in the anterior compartment. Thus, proper regulation of both the ci mRNA and protein appears to be critical for normal Drosophila development.     

Developmental Genetic Analysis of Contrabithorax Mutations in Drosophila Melanogaster

A developmental analysis of the Contrabithorax (Cbx) alleles offers the opportunity to examine the role of the Ultrabithorax (Ubx) gene in controlling haltere, as alternative to wing, morphogenesis in Drosophila. Several Cbx alleles are known with different spatial specificity in their wing toward haltere homeotic transformation. The molecular data on these mutations, however, does not readily explain differences among mutant phenotypes. In this work, we have analyzed the "apogenetic" mosaic spots of transformation in their adult phenotype, in mitotic recombination clones and in the spatial distribution of Ubx proteins in imaginal discs. The results suggest that the phenotypes emerge from early clonality in some Cbx alleles, and from cell-cell interactions leading to recruitment of cells to Ubx gene expression in others. We have found, in addition, mutual interactions between haltere and wing territories in pattern and dorsoventral symmetries, suggesting short distance influences, "accommodation," during cell proliferation of the anlage. These findings are considered in an attempt to explain allele specificity in molecular and developmental terms.     

Genetic requirements of vestigial in the regulation of Drosophila wing development

The gene vestigial has been proposed to act as a master gene because of its supposed capacity to initiate and drive wing development. We show that the ectopic expression of vestigial only induces ectopic outgrowths with wing cuticular differentiation and wing blade gene expression patterns in specific developmental and genetic contexts. In the process of transformation, wingless seems to be an essential but insufficient co-factor of vestigial. vestigial ectopic expression alone or vestigial plus wingless co-expression in clones differentiate 'mixed' cuticular patterns (they contain wing blade trichomes and chaetae characteristic of the endogenous surrounding tissue) and express wing blade genes only in patches of cells within the clones. In addition, we have found that these clones, in the wing imaginal disc, may cause autonomous as well as non-autonomous cuticular transformations and wing blade gene expression patterns. These non-autonomous effects in surrounding cells result from recruitment or 'inductive assimilation' of vestigial or wingless-vestigial overexpressing cells.     

The domineering non-autonomy of frizzled and van Gogh clones in the Drosophila wing is a consequence of a disruption in local signaling

The frizzled (fz) gene is required for the development of distally pointing hairs on the Drosophila wing. It has been suggested that fz is needed for the propagation of a signal along the proximal distal axis of the wing. The directional domineering non-autonomy of fz clones could be a consequence of a failure in the propagation of this signal. We have tested this hypothesis in two ways. In one set of experiments we used the domineering non-autonomy of fz and Vang Gogh (Vang) clones to assess the direction of planar polarity signaling in the wing. prickle (pk) mutations alter wing hair polarity in a cell autonomous way, so pk cannot be altering a global polarity signal. However, we found that pk mutations altered the direction of the domineering non-autonomy of fz and Vang clones, arguing that this domineering non-autonomy is not due to an alteration in a global signal. In a second series of experiments we ablated cells in the pupal wing. We found that a lack of cells that could be propagating a long-range signal did not alter hair polarity. We suggest that fz and Vang clones result in altered levels of a locally acting signal and the domineering non-autonomy results from wild-type cells responding to this abnormal signal.     

Genotoxicity testing of five herbicides in the Drosophila wing spot test

Four triazine herbicides: amitrole, metribuzin, prometryn and terbutryn, and the bipyridal compound diquat dibromide have been evaluated for genotoxicity in the wing somatic mutation and recombination test of Drosophila melanogaster, following standard procedures. Third-instar larvae trans-heterozygous for the third chromosome recessive markers multiple wing hairs (mwh) and flare-3 (flr(3)) were chronically fed with different concentrations of the test compounds. Feeding ended with pupation of the surviving larvae. Genetic changes induced in somatic cells of the wing's imaginal discs lead to the formation of mutant clones on the wing blade. Point mutation, chromosome breakage and mitotic recombination produce single spots; while twin spots are produced only by mitotic recombination. Exposure to 0.5 mM and 1 mM of amitrole clearly increased the frequency of small single, large single and total spots. Terbutryn, at the concentration of 5 mM, induced a slight increase in the frequency of small single and total spots, but this result could be false positive. The other three herbicides tested did not show any genotoxic effect. When heterozygous larvae for mwh and the multiple inverted TM3 balancer chromosomes were treated, significant increases in the frequency of mutant spots were only detected for amitrole. The observed spot frequencies were lower than those found in mwh/flr(3)50%) of the total spot induction was due to mitotic recombination.     

Drosophila Syntaxin Is Required for Cell Viability and May Function in Membrane Formation and Stabilization

The role of the Drosophila homologue of syntaxin-1A (syx) in neurotransmission has been extensively studied. However, developmental Northern analyses and in situ hybridization experiments show that SYX mRNA is expressed during all stages and in many tissues. We have isolated new mutations in syx that reveal roles for syx outside the nervous system. In the ovary, SYX is present in the germarium, but it is predominantly localized to nurse cell membranes. Mitotic recombination experiments in the germ-line show SYX is essential for oogenesis and may participate in membrane biogenesis in the nurse cells. In the early embryo, a large contribution of maternally deposited RNA is present, and the protein is localized at cell membranes during cellularization. After the maternal contribution is depleted, zygotically produced SYX assists secretion events occurring late in embryogenesis, such as cuticle deposition and neurotransmitter release. However, SYX is also required in larval imaginal discs, as certain hypomorphic mutant combinations exhibit rough eyes and wing notch defects indicative of cell death. Furthermore, recombinant clones that lack syx cause cell lethality in the developing eye. We propose that, similar to its roles in cuticle secretion and neurotransmitter release, SYX may mediate membrane assembly events throughout Drosophila development.     

Screening of larval/pupal P-element induced lethals on the second chromosome in Drosophila melanogaster: clonal analysis and morphology of imaginal discs

We have carried out screens for lethal mutations on the second chromosome of Drosophila melanogaster that are associated with abnormal imaginal disc morphologies, particularly in the wing disc. From a collection of 164 P element-induced mutations with a late larva/pupa lethal phase we have identified 56 new loci whose gene products are required for normal wing disc development and for normal morphology of other larval organs. Genetic mosaics of these 56 mutant lines show clonal mutant phenotypes for 23 cell-viable mutations. These phenotypes result from altered cell parameters. Causal relationships between disc and clonal phenotypes are discussed.     

Mutations at the fat locus interfere with cell proliferation control and epithelial morphogenesis in Drosophila

Lethal mutations at the fat locus in Drosophila cause imaginal discs to continue to grow by cell proliferation far beyond their normal final size. During a greatly extended larval period, the overgrowing imaginal discs develop additional folds and lobes, but retain a single-layered epithelial structure. In the wing disc, the additional lobes originate in the proximal fold area, and in the extra tissue the cells are less columnar than normal. Mutant disc cells lack zonulae adherents as well as associated microtubules and microfilaments, and they show an abnormal distribution and reduced density of gap junctions. The effect on growth is disc-autonomous as shown by transplantation experiments. The overgrown imaginal discs retain the ability to differentiate adult cuticular structures, as shown by metamorphosis of discs after transplantation into wild-type larval hosts and by the ability of some mutant animals to develop to the pharate adult stage. The structures differentiated by mutant discs show many abnormalities including ingrowths, outgrowths, separated cuticular vesicles, and areas of reversed bristle polarity; some of these abnormalities suggest that the mutations interfere with cell adhesion as well as the control of cell proliferation. The fat locus is located in cytogenetic interval 24D5.6-7, and 18 alleles are known including spontaneous, chemically induced, X-ray-induced, and dysgenic mutations.     

Mutations in the polycomb group gene polyhomeotic lead to epithelial instability in both the ovary and wing imaginal disc in Drosophila

Background

Most human cancers originate from epithelial tissues and cell polarity and adhesion defects can lead to metastasis. The Polycomb-Group of chromatin factors were first characterized in Drosophila as repressors of homeotic genes during development, while studies in mammals indicate a conserved role in body plan organization, as well as an implication in other processes such as stem cell maintenance, cell proliferation, and tumorigenesis. We have analyzed the function of the Drosophila Polycomb-Group gene polyhomeotic in epithelial cells of two different organs, the ovary and the wing imaginal disc.

Results

Clonal analysis of loss and gain of function of polyhomeotic resulted in segregation between mutant and wild-type cells in both the follicular and wing imaginal disc epithelia, without excessive cell proliferation. Both basal and apical expulsion of mutant cells was observed, the former characterized by specific reorganization of cell adhesion and polarity proteins, the latter by complete cytoplasmic diffusion of these proteins. Among several candidate target genes tested, only the homeotic gene Abdominal-B was a target of PH in both ovarian and wing disc cells. Although overexpression of Abdominal-B was sufficient to cause cell segregation in the wing disc, epistatic analysis indicated that the presence of Abdominal-B is not necessary for expulsion of polyhomeotic mutant epithelial cells suggesting that additional POLYHOMEOTIC targets are implicated in this phenomenon.

Conclusion

Our results indicate that polyhomeotic mutations have a direct effect on epithelial integrity that can be uncoupled from overproliferation. We show that cells in an epithelium expressing different levels of POLYHOMEOTIC sort out indicating differential adhesive properties between the cell populations. Interestingly, we found distinct modalities between apical and basal expulsion of ph mutant cells and further studies of this phenomenon should allow parallels to be made with the modified adhesive and polarity properties of different types of epithelial tumors.     

A screen to identify Drosophila genes required for integrin-mediated adhesion.

Drosophila integrins have essential adhesive roles during development, including adhesion between the two wing surfaces. Most position-specific integrin mutations cause lethality, and clones of homozygous mutant cells in the wing do not adhere to the apposing surface, causing blisters. We have used FLP-FRT induced mitotic recombination to generate clones of randomly induced mutations in the F1 generation and screened for mutations that cause wing blisters. This phenotype is highly selective, since only 14 lethal complementation groups were identified in screens of the five major chromosome arms. Of the loci identified, 3 are PS integrin genes, 2 are blistered and bloated, and the remaining 9 appear to be newly characterized loci. All 11 nonintegrin loci are required on both sides of the wing, in contrast to integrin alpha subunit genes. Mutations in 8 loci only disrupt adhesion in the wing, similar to integrin mutations, while mutations in the 3 other loci cause additional wing defects. Mutations in 4 loci, like the strongest integrin mutations, cause a "tail-up" embryonic lethal phenotype, and mutant alleles of 1 of these loci strongly enhance an integrin mutation. Thus several of these loci are good candidates for genes encoding cytoplasmic proteins required for integrin function.     

Defective gap-junctional communication associated with imaginal disc overgrowth and degeneration caused by mutations of the dco gene in Drosophila

The lethal(3)discs overgrown (dco) locus of Drosophila melanogaster, located on the third chromosome at cytogenetic position 100A5,6-100B1,2, is necessary for normal development and growth control in the imaginal discs of the larva. Three recessive lethal alleles (dco2, dco3, and dco18) in heteroallelic combinations and one allele (dco3) when homozygous cause the imaginal discs to continue to grow beyond the normal disc-intrinsic limit during an extended larval period. Some degeneration also occurs in the overgrowing discs. The discs overgrow even when transplanted early in their development into wild-type hosts, whereas normal discs stop growth at about the normal final size under such conditions, indicating that the overgrowth is a disc-autonomous effect of the mutations. During overgrowth the imaginal discs retain their single-layered epithelial structure except near regions of degeneration, and they differentiate into disc-appropriate but abnormal adult structures when transplanted into wild-type larval hosts. When the mutant larvae are reared under certain conditions a small percentage develop to the pharate adult stage, and these animals show a characteristic syndrome of abnormalities including swollen leg segments with many extra bristles, small or missing eyes, duplicated antennae and palpi, and separated vesicles of cuticle. A fourth recessive lethal allele (dcole88), when homozygous or in heteroallelic combination with the overgrowth alleles, causes the imaginal discs to degenerate, producing a "discless" phenotype. Gap junction-mediated communication was assayed by observing the intercellular transfer of injected fluorescein complexon (dye coupling). Dye coupling in the imaginal discs of the dco genotypes that cause overgrowth was dramatically reduced at 4 days after egg laying (AEL) compared with wild-type controls. Coupling was more normal although still significantly reduced at 7-8 and 12-14 days AEL. In c43hs1, another disc overgrowth mutant, the imaginal disc cells also showed very reduced dye coupling at 4 days and incomplete coupling at 9 days. In contrast, discs from wild-type larvae, two other imaginal disc overgrowth mutants, and a cell death mutant showed extensive dye coupling at all stages tested. Electron microscopic morphometry revealed a reduction in gap-junction length per unit lateral plasma membrane length in dco3/dco18 and c43hs1 wing discs, although not in dco2/dco3, compared with wild-type wing discs. The results suggest that gap-junctional cell communication may be involved in the cell interactions that limit cell proliferation in vivo.     

Genetic interactions between the RhoA and Stubble-stubbloid loci suggest a role for a type II transmembrane serine protease in intracellular signaling during Drosophila imaginal disc morphogenesis

The Drosophila RhoA (Rho1) GTPase is essential for postembryonic morphogenesis of leg and wing imaginal discs. Mutations in RhoA enhance leg and wing defects associated with mutations in zipper, the gene encoding the heavy chain of nonmuscle myosin II. We demonstrate here that mutations affecting the RhoA signaling pathway also interact genetically with mutations in the Stubble-stubbloid (Sb-sbd) locus that encodes an unusual type II transmembrane serine protease required for normal leg and wing morphogenesis. In addition, a leg malformation phenotype associated with overexpression of Sb-sbd in prepupal leg discs is suppressed when RhoA gene dose is reduced, suggesting that RhoA and Sb-sbd act in a common pathway during leg morphogenesis. We also characterized six mutations identified as enhancers of zipper mutant leg defects. Three of these genes encode known members of the RhoA signaling pathway (RhoA, DRhoGEF2, and zipper). The remaining three enhancer of zipper mutations interact genetically with both RhoA and Sb-sbd mutations, suggesting that they encode additional components of the RhoA signaling pathway in imaginal discs. Our results provide evidence that the type II transmembrane serine proteases, a class of proteins linked to human developmental abnormalities and pathology, may be associated with intracellular signaling required for normal development.