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.

GAL4/UAS targeted gene expression for studying Drosophila Hedgehog signaling.

The GAL4/upstream activating sequence (UAS) system is one of the most powerful tools for targeted gene expression. It is based on the properties of the yeast GAL4 transcription factor which activates transcription of its target genes by binding to UAS cis-regulatory sites. In Drosophila, the two components are carried in separate lines allowing for numerous combinatorial possibilities. The driver lines provide tissue-specific GAL4 expression and the responder lines carry the coding sequence for the gene of interest under the control of UAS sites. In this chapter, the basic GAL4/UAS system and its extensions, namely those allowing precise temporal control and reversible expression, are described. In addition, a list of GAL4 and UAS lines and schematic maps of GAL4 and UAS vectors useful in the study of Hedgehog (Hh) signaling is given. Finally, uses of the GAL4/UAS system to resolve some of the questions addressed in the study of the Hh pathway are presented.

fused regulates germline cyst mitosis and differentiation during Drosophila oogenesis.

The fused gene encodes a serine-threonine kinase that functions as a positive regulator of Hedgehog signal transduction in Drosophila embryogenesis, wing morphogenesis, and somatic cell development during oogenesis. Here, we have characterized the germline ovarian tumors present in adult ovaries of fused mutant females, a phenotype not observed upon deregulation of any other component of Hedgehog signaling. In the strongest fused mutant contexts, we found that tumorous ovarian follicles accumulate early spectrosome-containing germ cells corresponding to germline stem cells and/or early cystoblasts as evidenced by activated Dpp signal transduction and transcriptional repression of bag-of-marbles, encoding the cystoblast determination factor. These early germ cells are maintained far from their usual position in a specialized niche of somatic cells in the apical part of the germarium, which appears normal in size in fused mutant ovarioles. Therefore, these results indicate a novel function for fused in downregulation of Dpp signaling which is necessary for de-repression of bag-of-marbles and consequent cystoblast determination. The abnormal accumulation of these early germ cells seems to be due primarily to defects in differentiation since we show that germline stem cell proliferation in the germarium is not affected. A later block in germline development, at the 16-cell cyst stage before significant nurse cell and oocyte differentiation, was also observed in tumorous follicles when fused function was only partially lowered. Finally, fused mutant ovaries exhibit some germline cysts having undergone a supernumerary fifth mitotic division. Through clonal analysis, we provide evidence that fused regulates these cystocyte divisions cell autonomously, while the tumorous phenotype probably reflects both a somatic and germline requirement for fused for cyst and follicle development.

Modulation of the Suppressor of fused protein regulates the Hedgehog signaling pathway in Drosophila embryo and imaginal discs.

The Suppressor of fused (Su(fu)) protein is known to be a negative regulator of Hedgehog (Hh) signal transduction in Drosophila imaginal discs and embryonic development. It is antagonized by the kinase Fused (Fu) since Su(fu) null mutations fully suppress the lack of Fu kinase activity. In this study, we overexpressed the Su(fu) gene in imaginal discs and observed opposing effects depending on the position of the cells, namely a repression of Hh target genes in cells receiving Hh and their ectopic expression in cells not receiving Hh. These effects were all enhanced in a fu mutant context and were suppressed by cubitus interruptus (Ci) overexpression. We also show that the Su(fu) protein is poly-phosphorylated during embryonic development and these phosphorylation events are altered in fu mutants. This study thus reveals an unexpected role for Su(fu) as an activator of Hh target gene expression in absence of Hh signal. Both negative and positive roles of Su(fu) are antagonized by Fused. Based on these results, we propose a model in which Su(fu) protein levels and isoforms are crucial for the modulation of the different Ci states that control Hh target gene expression.

Hedgehog signaling controls Soma-Germen interactions during Drosophila ovarian morphogenesis.

The genetic analysis of Drosophila adult oogenesis has provided insights into the molecular mechanisms that control cell proliferation, differentiation, migration, and intercellular signaling. However, little is known about the larval and pupal cellular events leading to the formation of the highly organized adult ovary, which is composed of ovarioles each containing germline cells enveloped by specialized somatic cells. We describe here the presence of ovarioles devoid of any germ cells in adult females mutant for fused, which encodes a Hedgehog signal transducing serine/threonine kinase. We show that this phenotype corresponds to a requirement for fused function for the organization of germ cells with respect to ovarian somatic cells during ovariole formation specifically during pupal stages and provide some evidence by means of clonal analysis suggesting that fused function may be necessary in the germline. hedgehog is expressed specifically in somatic terminal filament cells in pupal ovaries, and females bearing hedgehog strong loss-of-function mutations also exhibit aberrant germ cell distribution and formation of agametic ovarioles. These results indicate a positive role for Fused in the transduction of somatic Hedgehog signaling instructing ovariole morphogenesis. We also provide evidence for the use of noncanonical Hedgehog signal transducer(s) within germline cells.

polyhomeotic is required for somatic cell proliferation and differentiation during ovarian follicle formation in Drosophila.

The polyhomeotic (ph) gene of Drosophila is a member of the Polycomb group (Pc-G) genes, which are required for maintenance of a repressed state of homeotic gene transcription, which stabilizes cell identity throughout development. The ph gene was recovered in the course of a gain-of-function screen aimed at identifying genes with a role during ovarian follicle formation in Drosophila, a process that involves coordinated proliferation and differentiation of two cell lineages, somatic and germline. Subsequent analysis revealed that ph loss-of-function mutations lead to production of follicles with greater or fewer than the normal number of germ cells associated with reduced proliferation of somatic prefollicular cells, abnormal prefollicular cell encapsulation of germline cysts and an excess of both interfollicular stalk cells and polar cells. Clonal analysis showed that ph function for follicle formation resides specifically in somatic cells and not in the germline. This is thus the first time that a role has been shown for a Pc-G gene during Drosophila folliculogenesis. In addition, we tested mutations in a number of other Pc-G genes, and two of them, Sex combs extra (Sce) and Sex comb on midleg (Scm), also displayed ovarian defects similar to those observed for ph. Our results provide a new model system, the Drosophila ovary, in which the function of Pc-G genes, distinct from that of control of homeotic gene expression, can be explored.

Fused-dependent Hedgehog signal transduction is required for somatic cell differentiation during Drosophila egg chamber formation.

The fused gene encodes a serine/threonine kinase involved in Hedgehog signal transduction during Drosophila embryo and larval imaginal disc development. Additionally, fused mutant females exhibit reduced fecundity that we report here to be associated with defects in three aspects of egg chamber formation: encapsulation of germline cysts by prefollicular cells in the germarium, interfollicular stalk morphogenesis and oocyte posterior positioning. Using clonal analysis we show that fused is required cell autonomously in prefollicular and pre-stalk cells to control their participation in these aspects of egg chamber formation. In contrast to what has been found for Hedgehog and other known components of Hedgehog signal transduction, we show that fused does not play a role in the regulation of somatic stem cell proliferation. However, genetic interaction studies, as well as the analysis of the effects of a partial reduction in Hedgehog signaling in the ovary, indicate that fused acts in the classical genetic pathway for Hedgehog signal transduction which is necessary for somatic cell differentiation during egg chamber formation. Therefore, we propose a model in which Hedgehog signals at least twice in germarial somatic cells: first, through a fused-independent pathway to control somatic stem cell proliferation; and second, through a classical fused-dependent pathway to regulate prefollicular cell differentiation.

Genetic analysis of viable and lethalfused mutants ofDrosophila melanogaster.

Fused is a segmentation gene belonging to the segment-polarity class. Mutations at thefused locus are known to display pleiotropic effects, causing zygotically determined anomalies of ovaries and of some adult cuticular structures, and maternally determined embryonic segmentation defects. In order to determine the amorphic phenotype offused and to study the genetical basis of its pleiotropy, newfused alleles (18 viable and 11 lethal) were isolated. The phenotype of these mutants and of others already known are described, taking into account zygotic and maternal effects. The main results provided by this analysis are as follows. Firstly, allfused alleles show the whole complex fused phenotype, and a good correlation is observed between the strength of the wing and segmentation defects, suggesting that a single function is involved in both processes. Secondly, all embryonic and larval lethals carry deficiencies which allow us to localizefused between the 17C4 and 17D2 bands of the X-chromosome. Thirdly, the 24 viable and 2 pupal lethals examined behave as point mutants, as shown cytologically or by Southern blot analysis. However, only one of them, the pupal lethalfu mH63 was proven to carry a nullfused allele, since it displays in germ-line clones a strong maternal phenotype and a very low zygotic rescue, similar to those of the small deficiencyDf(1)fu z4. The phenotype of the amorphic mutant indicates that zygotic ezpression offused is required for normal metamorphosis, while maternal expression is necessary for a normal segmentation pattern, since a complete loss offused expression during oogenesis cannot be compensated zygotically.