Regulation of cellular plasticity in Drosophila imaginal disc cells by the Polycomb group, trithorax group and lama genes

Drosophila imaginal disc cells can switch fates by transdetermining from one determined state to another. We analyzed the expression profiles of cells induced by ectopic Wingless expression to transdetermine from leg to wing by dissecting transdetermined cells and hybridizing probes generated by linear RNA amplification to DNA microarrays. Changes in expression levels implicated a number of genes: lamina ancestor, CG12534 (a gene orthologous to mouse augmenter of liver regeneration), Notch pathway members, and the Polycomb and trithorax groups of chromatin regulators. Functional tests revealed that transdetermination was significantly affected in mutants for lama and seven different PcG and trxG genes. These results validate our methods for expression profiling as a way to analyze developmental programs, and show that modifications to chromatin structure are key to changes in cell fate. Our findings are likely to be relevant to the mechanisms that lead to disease when homologs of Wingless are expressed at abnormal levels and to the manifestation of pluripotency of stem cells.     

Ectopic expression of BEAF32A in the Drosophila eye imaginal disc inhibits differentiation of photoreceptor cells and induces apoptosis

Transgenic flies were established in which ectopic expression of boundary element-associated factor (BEAF) 32A was targeted to the Drosophila eye imaginal disc. The eyes of the adult fly displayed a severe rough eye phenotype. When these eyes were sectioned, most ommatidia were found to be fused and irregularly shaped rhabdomeres were observed. In the developing eye imaginal disc, expression of BEAF32A inhibited differentiation of photoreceptor cells. Expression of BEAF32A also induced extensive apoptosis of eye imaginal disc cells and, consistent with this, co-expression of baculovirus P35 in the eye imaginal disc suppressed the BEAF32A-induced rough eye phenotype. To investigate the effects of BEAF32A on regulation of chromatin structure, genetic crosses of the BEAF32A-overexpressing flies with loss-of-function mutants for genes encoding other boundary element-binding factors or regulators of chromatin structure were conducted. Interestingly, half-dose reduction of the su(Hw) gene strongly enhanced the rough eye phenotype induced by BEAF32A. Furthermore, genetic crosses of the transgenic flies with loss-of-function mutants for genes interacting with Polycomb revealed specific links between BEAF32A and genes such as Distalless and kohtalo, suggesting a relation to the chromatin insulator function of BEAF. In addition, genetic crosses of transgenic flies expressing BEAF32A with a collection of Drosophila deficiency stocks allowed us to identify several genomic regions, deletions of which caused enhancement or suppression of the BEAF32A-induced rough eye phenotype. The transgenic flies established in this study should be useful to identify targets of BEAF32A and its positive or negative regulators in Drosophila.     

Over-expression of transglutaminase in the Drosophila eye imaginal disc induces a rough eye phenotype

Transglutaminases (TGs) catalyze the cross-linking of proteins and are involved in various biological processes in mammals. In invertebrates, except for the involvement in the hemolymph clotting, the functions of TG have not been revealed. Drosophila has a single TG gene (CG7356), from which two kinds of mRNAs (dTG-RA and dTG-RB) are formed. RT-PCR analyses indicated that both dTGs-RA and -RB are synthesized in all the developmental stages tested. To reveal the roles of dTG during the development, we examined a phenotype induced through the ectopic expression of dTG by using a GAL4-UAS targeted expression system. Over-expression of dTG-A in the eye imaginal disc of larva induced a rough eye phenotype in adult compound eyes. Co-expression of P35, an inhibitor of apoptosis, suppressed the rough eye phenotype, suggesting that the rough eye phenotype induced by the over-expression of dTG-A in the eye imaginal disc is due to the occurrence of apoptosis. The rough eye phenotype induced by the over-expression of dTG-A was suppressed by the crossing with mutant fly lines lacking Drosophila JNK gene basket (bsk) or Drosophila JNKK gene hemipterous. FLP-out experiments using an enhancer trap line showed that the over-expression of dTG-A in the eye imaginal disc increased the puckered enhancer activity, a reporter of Bsk activity. These results suggested that the rough eye phenotype induced by the over-expression of dTG-A is related to an enhancement of JNK signaling pathway.     

Ectopic expression of homeotic genes caused by the elimination of the Polycomb gene in Drosophila imaginal epidermis

The morphological patterns in the adult cuticle of Drosophila are determined principally by the homeotic genes of the bithorax and Antennapedia complexes. We find that many of these genes become indiscriminately active in the adult epidermis when the Pc gene is eliminated. By using the Pc3 mutation and various BX-C mutant combinations, we have generated clones of imaginal cells possessing different combinations of active homeotic genes. We find that, in the absence of BX-C genes, Pc- clones develop prothoracic patterns; this is probably due to the activity of Sex combs reduced which overrules Antennapedia. Adding contributions of Ultrabithorax, abdominal-A and Abdominal-B results in thoracic or abdominal patterns. We have established a hierarchical order among these genes: Antp less than Scr less than Ubx less than abd-A less than Abd-B. In addition, we show that the engrailed gene is ectopically active in Pc- imaginal cells.     

Genomic imprinting in Drosophila is maintained by the products of Suppressor of variegation and trithorax group,but not Polycomb group,genes

Genomic imprinting is a form of epigenetic inheritance that is characterized by differential expression of a gene depending on its parental origin. The mini-X chromosome Dp(1;f)LJ9 in Drosophila shows this type of classical imprinting; when transmitted by the maternal parent genes on this chromosome are fully expressed, but when the chromosome is transmitted by the male parent at least three genes are subject to silencing, resulting in a variegated expression pattern. Chemical and environmental modifiers of position-effect variegation have been shown to alter the somatic maintenance of the imprint. To extend these observations, several mutations in chromatin-associated proteins were examined for their effect on imprinting on the Dp(1;f)LJ9 mini-X chromosome. Effects on establishment and maintenance were independently assessed by genetically associating the mutations in chromatin modifiers with the mini-X chromosome in either the parents, where the imprint is established, or the progeny, in which the imprint must be maintained. Nine Suppressor of variegation [ Su(var)] mutations, including alleles of the Su(var)2-5 gene, which encodes the well characterized heterochromatin-associated protein HP1, abolished maintenance but not the establishment of the imprint. Mutant alleles of two genes in the trithorax group ( trx-G), brahma and trithorax, showed a maternal-effect enhancement of the paternal imprint. Surprisingly, however, with the exception of an Enhancer of Polycomb [ E(Pc)] allele, none of the Polycomb-group ( Pc-G) mutations tested affected the imprint. Thus, the maintenance of this imprint relies on the wild-type products of Su(var) and trx-G, but not Pc-G, genes. Finally, none of the mutations tested affected the maintenance of the maternal imprint or the establishment of either the maternal or paternal imprint, suggesting that the maternal and paternal imprints depend on different molecular processes and that imprint establishment and maintenance are independently regulated.     

patufet, the gene encoding the Drosophila melanogaster homologue of selenophosphate synthetase, is involved in imaginal disc morphogenesis

Proliferation in imaginal discs requires cell growth and is linked to patterning processes controlled by secreted cell-signalling molecules. To identify new genes involved in the control of cell proliferation we have screened a collection of P-lacW insertion mutants that result in lethality in the larval/pupal stages, and characterized a novel gene, patufet (ptuf). Inactivation of ptuf by a P element insertion in the 5′ untranslated region leads to aberrant imaginal disc morphology characterized by a reduction in mass of discs and disorganisation of disc cells where no folding or patterning can be detected. Moreover, apoptotic cells can be observed in these small and abnormal mutant discs. To examine the role of ptuf we have studied its clonal behaviour in genetic mosaics generated by mitotic recombination. The mutation causes reduced cell viability, smaller cell size and stops vein differentiation. Non-autonomous effects, such as abnormal differentiation of wild-type cells surrounding the clones, are also observed. We have cloned the ptuf gene of Drosophila melanogaster and found that it encodes a selenophosphate synthetase, which is the first identified in insects. Mutant flies transformed with the full-length cDNA show complete reversion of lethality and disc phenotype. Northern blot analysis and in situ hybridization indicate that the ptuf gene is expressed in imaginal discs as well as at different stages of development. The synthesis of selenoproteins by the selenophosphate synthetase, the role of selenoproteins in the maintenance of the oxidant/antioxidant balance of the cell and its possible implications in imaginal disc morphogenesis are discussed.     

Cell proliferation and DNA replication in the imaginal wing disc of Drosophila melanogaster

Cell proliferation in the imaginal wing disc of Drosophila has been analyzed by both pulse and chronic labeling with [3H]thymidine. We find neither spatial nor temporal variation in the fraction of S phase cells during the third instar. At or near the time of white prepupae formation the fraction of S phase cells falls sharply. Our chronic labeling experiments have demonstrated that almost all (and perhaps all) of the cells in a mid third instar wing disc are cycling. By examining sectioned material from such experiments we have found that the collumnar epithelial cell and the adepithetial cell populations become labeled with similar kinetics. The peripodial membrane cell population becomes labeled more slowly. We have also obtained estimates of cell cycle parameters for the imaginal wing disc cells.     

Ectopic expression of dE2F and dDP induces cell proliferation and death in the Drosophila eye.

The deregulation of E2F activity is thought to contribute to the uncontrolled proliferation of many tumor cells. While the effects of overexpressing E2F genes have been studied extensively in tissue culture, the consequences of elevating E2F activity in vivo are unknown. To address this issue, transgenic lines of Drosophila were studied in which ectopic expression of dE2F and dDP was targeted to the developing eye. The co-expression of dDP or dE2F disrupted normal eye development, resulting in abnormal patterns of bristles, cone cells and photoreceptors. dE2F/dDP expression caused ectopic S phases in post-mitotic cells of the eye imaginal disc but did not disrupt the onset of neuronal differentiation. Most S phases were seen in uncommitted cells, although some cells that had initiated photo-receptor differentiation were also driven into the cell cycle. Elevated expression of dE2F and dDP caused apoptosis in the eye disc. The co-expression of baculovirus p35 protein, an inhibitor of cell death, strongly enhanced the dE2F/dDP-dependent phenotype. These results show that, in this in vivo system, the elevation of E2F activity caused post-mitotic cells to enter the cell cycle. However, these cells failed to proliferate unless rescued from apoptosis.     

Localization of the sevenless protein, a putative receptor for positional information, in the eye imaginal disc of Drosophila

The Drosophila gene sevenless encodes a putative trans-membrane receptor required for the formation of one particular cell, the R7 photoreceptor, in each ommatidium of the compound eye. Mutations in this gene result in the cell normally destined to form the R7 cell forming a non-neuronal cell type instead. These observations have led to the proposal that the sevenless protein receives at least part of the positional information required for the R7 developmental pathway. We have generated antibodies specific for sevenless and have examined expression of the protein by light and electron microscopy. sevenless protein is present transiently at high levels in at least 9 cells in each developing ommatidium and is detectable several hours before any overt differentiation of R7. The protein is mostly localized at the apices of the cells, in microvilli, but is also found deeper in the tissue where certain cells contact the R8 cell. This finding suggests that R8 expresses a ligand for the sevenless protein.     

Epigenetic inheritance of expression states in plant development: the role of Polycomb group proteins

Polycomb group (PcG) proteins maintain a repressed state of gene expression over many cell divisions. The recent characterisation of several PcG proteins from plants revealed a remarkable structural and functional conservation of PcG proteins between different kingdoms. In both plants and animals, homeotic genes are among the target genes of PcG complexes, although the structure of these genes is not conserved. However, not all PcG proteins identified in animals are present in plants. Furthermore it becomes clear that PcG-mediated repression in plants is more transient compared with the long-lasting effects in animals. This may be related to the absence of PcG proteins thought to be involved in long-term maintenance of PcG repression, suggesting that the mechanisms underlying PcG-mediated repression differ between plants and animals.