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Epithelial morphogenesis is marked by dynamic changes in cell sizes until the final adult organ is formed. Using a combination of quantitative, mathematical simulation and genetic techniques, this paper reveals a novel mechanism of epithelial cell size regulation based on heterophilic cell–cell adhesion between two atypical cadherins, Ft and Ds.

Huntington’s disease (HD) is a neurodegenerative disorder induced by aggregation of the pathological form of Huntingtin protein that has expanded polyglutamine (polyQ) repeats. In the Drosophilamodel, for instance, expression of transgenes with polyQ repeats induces HD-like pathologies, progressively correlating with the increasing lengths of these repeats. Previous studies on both animal models and clinical samples have revealed metabolite imbalances during HD progression. To further explore the physiological processes linked to metabolite imbalances during HD, we have investigated the 1D 1H NMR spectroscopy-based metabolomics profile of DrosophilaHD model. Using multivariate analysis (PCA and PLS-DA) of metabolites obtained from methanolic extracts of fly heads displaying retinal deformations due to polyQ overexpression, we show that the metabolite imbalance during HD is likely to affect cell energetics. Six out of the 35 metabolites analyzed, namely, nicotinamide adenine dinucleotide (NAD), lactate, pyruvate, succinate, sarcosine, and acetoin, displayed segregation with progressive severity of HD. Specifically, HD progression was seen to be associated with reduction in NAD and increase in lactate-to-pyruvate ratio. Furthermore, comparative analysis of fly HD metabolome with those of mouse HD model and HD human patients revealed comparable metabolite imbalances, suggesting altered cellular energy homeostasis. These findings thus raise the possibility of therapeutic interventions for HD via modulation of cellular energetics.

During tissue closures, such as embryonic dorsal closure in Drosophila melanogaster, a proximate extra-embryonic layer, amnioserosa, generates forces that drive migration of the flanking lateral embryonic epidermis, thereby zip-shutting the embryo. Arguably, this paradigm of tissue closure is also recapitulated in mammalian wound healing wherein proximate fibroblasts transform into contractile myofibroblasts, develop cell junctions, and form a tissue layer de novo: contraction of the latter then aids in wound closure. Given this parallelism between disparate exemplars, we posit a general principle of tissue closure via proximate cell layer-generated forces. Here, we have tested this hypothesis in pupal thorax closure wherein 2 halves of the presumptive adult thorax of Drosophila, the contralateral heminotal epithelia, migrate over an underlying larval epidermal cell layer. We show that the proximate larval epidermal cell layer promotes thorax closure by its active contraction, orchestrated by its elaborate actomyosin network-driven epithelial cell dynamics, cell delamination, and death—the latter being prefigured by the activation of caspases. Larval epidermal cell dynamics generate contraction forces, which when relayed to the flanking heminota—via their mutual integrin-based adhesions—mediate thorax closure. Compromising any of these contraction force-generating mechanisms in the larval epidermal cell layer slows down heminotal migration, while loss of its relay to the flanking heminota abrogates the thorax closure altogether. Mathematical modeling further reconciles the biophysical underpinning of this emergent mechanism of thorax closure. Revealing mechanism of thorax closure apart, these findings show conservation of an essential principle of a proximate cell layer-driven tissue closure.

TFIID is a multiprotein complex composed of the TATA binding protein (TBP) and TBP-associated factors (TAFIIs). The binding of TFIID to the promoter is the first step of RNA polymerase II preinitiation complex assembly on protein-coding genes. Yeast (y) and human (h) TFIID complexes contain 10 to 13 TAFIIs. Biochemical studies suggested that the Drosophila(d) TFIID complexes contain only eight TAFIIs, leaving a number of yeast and human TAFIIs (e.g., hTAFII55, hTAFII30, and hTAFII18) without knownDrosophilahomologues. We demonstrate thatDrosophilahas not one but two hTAFII30 homologues, dTAFII16 and dTAFII24, which are encoded by two adjacent genes. These two genes are localized in a head-to-head orientation, and their 5′ extremities overlap. We show that these novel dTAFIIs are expressed and that they are both associated with TBP and other bona fide dTAFIIs in dTFIID complexes. dTAFII24, but not dTAFII16, was also found to be associated with the histone acetyltransferase (HAT) dGCN5. Thus, dTAFII16 and dTAFII24 are functional homologues of hTAFII30, and this is the first demonstration that a TAFII-GCN5-HAT complex exists in Drosophila. The two dTAFIIs are differentially expressed during embryogenesis and can be detected in both nuclei and cytoplasm of the cells. These results together indicate that dTAFII16 and dTAFII24 may have similar but not identical functions.

ABSTRACTTFIID is a multiprotein complex composed of the TATA binding protein (TBP) and TBP-associated factors (TAFIIs). The binding of TFIID to the promoter is the first step of RNA polymerase II preinitiation complex assembly on protein-coding genes. Yeast (y) and human (h) TFIID complexes contain 10 to 13 TAFIIs. Biochemical studies suggested that the Drosophila(d) TFIID complexes contain only eight TAFIIs, leaving a number of yeast and human TAFIIs (e.g., hTAFII55, hTAFII30, and hTAFII18) without knownDrosophilahomologues. We demonstrate thatDrosophilahas not one but two hTAFII30 homologues, dTAFII16 and dTAFII24, which are encoded by two adjacent genes. These two genes are localized in a head-to-head orientation, and their 5' extremities overlap. We show that these novel dTAFIIs are expressed and that they are both associated with TBP and other bona fide dTAFIIs in dTFIID complexes. dTAFII24, but not dTAFII16, was also found to be associated with the histone acetyltransferase (HAT) dGCN5. Thus, dTAFII16 and dTAFII24 are functional homologues of hTAFII30, and this is the first demonstration that a TAFII-GCN5-HAT complex exists inDrosophila. The two dTAFIIs are differentially expressed during embryogenesis and can be detected in both nuclei and cytoplasm of the cells. These results together indicate that dTAFII16 and dTAFII24 may have similar but not identical functions.

Growth and patterning during Drosophilawing development are mediated by signaling from its dorsoventral (D/V) organizer. In the metathorax, wing development is essentially suppressed by the homeotic selector gene Ultrabithorax(Ubx) to mediate development of a pair of tiny balancing organs, the halteres. Here we show that expression of Ubxin the haltere D/V boundary down-regulates its D/V organizer signaling compared to that of the wing D/V boundary. Somatic loss of Ubxfrom the haltere D/V boundary thus results in the formation of a wing-type D/V organizer in the haltere field. Long-distance signaling from this organizer was analyzed by assaying the ability of a Ubx−clone induced in the haltere D/V boundary to effect homeotic transformation of capitellum cells away from the boundary. The clonally restored wing D/V organizer in mosaic halteres not only enhanced the homeotic transformation of Ubx−cells in the capitellum but also caused homeotic transformation of even Ubx+cells in a genetic background known to induce excessive cell proliferation in the imaginal discs. In addition to demonstrating a non-cell-autonomous role for Ubxduring haltere development, these results reveal distinct spatial roles of Ubxduring maintenance of cell fate and patterning in the halteres.

Homozygosity forlethal(2)giant larvae (lgl),a mutation in a tumor suppressor gene ofDrosophila,induces neoplasia of the imaginal discs. To explore the developmental capacities oflglmutant cells, we have investigated their growth and differentiation in genetic mosaics. Adult wings mosaic forlgldisplayed abnormal growth and differentiation of thelglmutant and neighboring wild-type cells, suggesting aberrant cell–cell interactions during development.lglmutant clones also straddled 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 oflglmutant larvae, we studied the expression of anengrailed (en)-specificlacZreporter gene during progressive stages of their tumorous growth. Our results show thatenis activated in the posterior compartments of the neoplastic imaginal discs. However, during later stages of tumorous overgrowth, theen-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.

Developmental expression of transduced mini-white(w) gene of Drosophilais sensitive to its flanking genomic enhancers. Taking advantage of this phenomenon, we mobilized a P lacWtransposon and screened for new transposant lines which showed patterned expression of the mini-w gene in adult eyes. From a screen of about 1,000 independent P lacWtransposant lines on the second chromosome, we identified 7 lines which showed patterned w expression in adult eyes. These P insertions were assigned to engrailed, winglessand teashirtgenes based on their chromosomal locations, developmental expression of the lacZreporter gene, lethal embryonic mutant phenotypes and, finally, their failure to complement the lethal alleles of the respective genetic loci. Our results show that although only a small fraction of the total transposant lines displayed patterned w expression, the genetic loci thus identified are those which play essential roles in pattern formation. Scopes of screens for genetic loci based on wreporter gene expression in adult eyes are discussed.

TheDrosophilahomologue (D-abl) of the mammalianabelsonproto-oncogene (c-abl) encodes a cytoplasmic protein tyrosine kinase which localizes to axons of the developing embryonic central nervous system (CNS) and has been shown to be required for redundant functions during axonogenesis. These redundant functions become indispensable when other components of the redundant pathway, such as those encoded bydisabled(dab), fasciclin I (fas I) orfailed axon connection(fax) are removed fromablmutants. Second-site mutations can thus uncover redundant aspects ofabl-mediated axonogenesis. We used this strategy, and present evidence to suggest a redundant function of the cytoskeletal protein encoded by thelethal(2)giant larvae(lgl) tumour suppressor gene during embryonic axonogenesis. Simultaneous mutation inlglandablshifts lethality of the mutations to late embryogenesis while mutation in only one of these genes permits development up to late larval/pupal or pharate adult stages. Thelgl-;abl-embryos show defective development of the CNS, characterized by loss of axonal commissures and longitudinal axonal tracts. Lethality of the double mutation is aggravated or suppressed bydisabled(dab) orenabled(ena) mutations, which act, respectively, as dominant enhancers or suppressors ofabl. The redundant function oflgltumour suppressor gene during axonogenesis therefore appears to involve aspects of D-abl-mediated signalling.

Lethal mutations in the giant discs (lgd) and fat (ft) tumor suppressor genes of Drosophila cause epithelial hyperplasia in all imaginal discs. By contrast, mutations in the vestigial (vg) gene adversely affect cell viability in the wing imaginal discs and consequently cause loss of pattern in the adult wings. However, combining homozygous lgd or ft mutations with homozygous vg¹ increases the size of the wing imaginal discs and partially restores the bristle pattern in the wings of pharate adults. Comparable pattern restoration in vg¹ wings is also induced by a newly isolated weak hypomorphic lgd³ allele. Further, mosaic analysis revealed that whereas lgd clones generated by the Minute technique display abnormal differentiation, those induced in a homozygous vg¹ background exhibit autonomous restoration of wing pattern. These results suggest that pattern restoration in vg¹ wings can serve as an assay for hyperplasia induced by mutations in Drosophila tumor suppressor genes. Copyright 1995, 1999 Academic Press

Homozygosity for recessive mutations inDrosophilatumour suppressor genes likelethal giant larvae (Igl), lethal giant discs (Igd)orfat (ft)induce uncontrolled cell proliferations in the imaginal discs of the mutant larvae. Imaginal discs of larvae mutant forIgltumour suppressor gene display neoplastic growths while those mutant forIgdorfatdisplay hyperplastic growths. Results presented in this study reveal that mutant wing imaginal discs with neoplastic or hyperplastic overgrowths display high mitotic activity primarily during the extended period of larval life when their wild-type siblings have already pupariated. Both these categories of overgrowths show overall stability of the karyotypes and only low frequency of aneuploidy. The hyperplastic imaginal discs ofIgdorftmutant larvae displayed normal chromosome condensation. In contrast, the neoplastic imaginal discs ofIglmutants showed high frequency of mitotic cells with undercondensed chromosomes. In this respect the neoplastic discs resemble malignant neuroblastomas of theIgllarvae which also display undercondensed chromosomes. These results thus suggest an indirect role of the cytoskeletal protein encoded byIgltumour suppressor gene in aspects of normal chromosome condensation during mitosis.

Peptide therapeutics, unlike small-molecule drugs, display crucial advantages of target specificity and the ability to block large interacting interfaces, such as those of transcription factors. The transcription co-factor of the Hippo pathway, YAP/Yorkie (Yki), has been implicated in many cancers, and is dependent on its interaction with the DNA-binding TEAD/Sd proteins via a large Ω-loop. In addition, the mammalian vestigial-like (VGLL) proteins, specifically their TONDU domain, competitively inhibit YAP-TEAD interaction, resulting in arrest of tumor growth. Here, we show that overexpression of the TONDU peptide or its oral uptake leads to suppression of Yki-driven intestinal stem cell tumors in the adult Drosophila midgut. In addition, comparative proteomic analyses of peptide-treated and untreated tumors, together with chromatin immunoprecipitation analysis, reveal that integrin pathway members are part of the Yki-oncogenic network. Collectively, our findings establish Drosophila as a reliable in vivo platform to screen for cancer oral therapeutic peptides and reveal a tumor suppressive role for integrins in Yki-driven tumors. This article has an associated First Person interview with the first author of the paper.

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