Crosstalk between chromatin and Shavenbaby defines transcriptional output along the Drosophila intestinal stem cell lineage.

The transcription factor Shavenbaby (Svb), the only member of the OvoL family in Drosophila, controls the fate of various epithelial embryonic cells and adult stem cells. Post-translational modification of Svb produces two protein isoforms, Svb-ACT and Svb-REP, which promote adult intestinal stem cell renewal or differentiation, respectively. To define Svb mode of action, we used engineered cell lines and develop an unbiased method to identify Svb target genes across different contexts. Within a given cell type, Svb-ACT and Svb-REP antagonistically regulate the expression of a set of target genes, binding specific enhancers whose accessibility is constrained by chromatin landscape. Reciprocally, Svb-REP can influence local chromatin marks of active enhancers to help repressing target genes. Along the intestinal lineage, the set of Svb target genes progressively changes, together with chromatin accessibility. We propose that Svb-ACT-to-REP transition promotes enterocyte differentiation of intestinal stem cells through direct gene regulation and chromatin remodeling.

[OvoL factors: a family of key regulators of epithelium mesenchyme plasticity and stem cells]. / Les facteurs OvoL - Des régulateurs clés de la plasticité épithélium-mésenchyme et des cellules souches.

Most prevalent cancers are of epithelial origin and their morbidity often results from secondary tumors. Cancer aggressiveness relates to intratumoral heterogeneity, including rare tumor initiating cells that share many features with adult stem cells. Both normal and cancer stem cells are characterized by their plasticity between epithelial and mesenchymal phenotypes, progressing through a series of reversible intermediates. While a core of regulators (Snail, Zeb1-2,...) is renowned to promote epithelial to mesenchyme transition (EMT), OvoL/Shavenbaby factors now emerge as a family of key epithelial stabilizers. Therefore, pro-EMT and OvoL/Shavenbaby transcription factors could provide a molecular rheostat to control stemness and epithelial-mesenchyme plasticity. We address this question in flies, in which the unique OvoL/Shavenbaby factor offers a powerful in vivo paradigm for functional analyses. Our results show that Shavenbaby is critical for adult stem cell homeostasis, and directly interacts with the Hippo pathway to protect stem cells from death.

TITLE: Les facteurs OvoL - Des régulateurs clés de la plasticité épithélium-mésenchyme et des cellules souches. ABSTRACT: Des avancées majeures révèlent l'hétérogénéité intra-tumorale des cancers d'origine épithéliale, incluant des cellules initiatrices de tumeurs qui ressemblent aux cellules souches adultes. Les cellules souches normales et tumorales partagent en effet leur plasticité entre phénotypes épithéliaux et mésenchymateux, progressant par une série d'états intermédiaires, réversibles. Si un cœur de régulateurs (Snail, Zeb, …) est bien connu pour déclencher la transition épithélio-mésenchymateuse (TEM), les facteurs OvoL/Shavenbaby sont récemment apparus comme des stabilisateurs épithéliaux. La balance entre facteurs pro-TEM et OvoL pourrait ainsi réguler la plasticité phénotypique et le potentiel métastatique des tumeurs. Nous abordons cette question chez la drosophile, un modèle pour disséquer in vivo la fonction de Shavenbaby. Nos travaux montrent que Shavenbaby est un régulateur clé de l'homéostasie des cellules souches adultes. Shavenbaby est indispensable à leur survie, agissant en interaction directe avec la voie Hippo pour protéger les cellules souches de la mort cellulaire programmée.

Hypergravity increases resistance to heat in dFOXO Drosophila melanogaster mutants and can lower FOXO translocation in wild-type males.

Severe stresses have deleterious effects, but mild stresses can have beneficial effects called hormetic effects. This study observed survival time of wild-type Drosophila melanogaster flies and dFOXO mutants exposed to 37 °C, a severe stress for flies, after they lived or not for 2 weeks in hypergravity (3 or 5 g), a mild stress with hormetic effects in flies. Hypergravity increased survival time of the mutants, this effect being less observed in wild-type flies. The heat stress increased dFOXO translocation similarly in all gravity groups in a wild-type strain, and hypergravity decreased dFOXO translocation similarly in heat-stressed or not heat-stressed males, no clear effect of the gravity level being observed in females. Because hypergravity increases resistance to heat in dFOXO mutants and the translocation is not tightly dependent on the gravity level, one can conclude that dFOXO does not mediate the effect of hypergravity on resistance to heat. A previous study showed that another mild stress, the cold, can increase survival time at 37 °C of wild-type D. melanogaster flies, but this was not observed in dFOXO mutants. Therefore, two mild stresses, cold and hypergravity, can increase resistance to heat but the pathways mediating this effect are seemingly different, as cold does not increase resistance in dFOXO mutants while hypergravity increases it.

Shavenbaby and Yorkie mediate Hippo signaling to protect adult stem cells from apoptosis.

To compensate for accumulating damages and cell death, adult homeostasis (e.g., body fluids and secretion) requires organ regeneration, operated by long-lived stem cells. How stem cells can survive throughout the animal life remains poorly understood. Here we show that the transcription factor Shavenbaby (Svb, OvoL in vertebrates) is expressed in renal/nephric stem cells (RNSCs) of Drosophila and required for their maintenance during adulthood. As recently shown in embryos, Svb function in adult RNSCs further needs a post-translational processing mediated by the Polished rice (Pri) smORF peptides and impairing Svb function leads to RNSC apoptosis. We show that Svb interacts both genetically and physically with Yorkie (YAP/TAZ in vertebrates), a nuclear effector of the Hippo pathway, to activate the expression of the inhibitor of apoptosis DIAP1. These data therefore identify Svb as a nuclear effector in the Hippo pathway, critical for the survival of adult somatic stem cells.

A mild cold stress that increases resistance to heat lowers FOXO translocation in Drosophila melanogaster.

Previous studies have shown that subjecting Drosophila melanogaster flies to a mild stress at young or middle age can increase lifespan and resistance to severe stresses throughout life and that the NF-κB-like transcription factor DIF, the 70 kDa heat-shock proteins, and the Drosophila Forkhead box class O (dFOXO) transcription factor could explain some of these effects. The present study showed that two dFOXO mutants do not survive longer heat if previously subjected to a mild cold stress, contrarily to wild-type flies. This cold pretreatment had nearly no effect on dFOXO nuclear translocation in wild-type males. Heat stress strongly increased dFOXO translocation, but this effect was lowered in cold-pretreated males. Because cold-pretreated wild-type males survived longer heat and had nevertheless a lower dFOXO translocation after this heat stress, one can conclude that dFOXO is required to resist heat but that the cold pretreatment makes that other mechanisms partly substitute to dFOXO translocation.

The EBF transcription factor Collier directly promotes Drosophila blood cell progenitor maintenance independently of the niche.

The maintenance of stem or progenitor cell fate relies on intrinsic factors as well as local cues from the cellular microenvironment and systemic signaling. In the lymph gland, an hematopoietic organ in Drosophila larva, a group of cells called the Posterior Signaling Centre (PSC), whose specification depends on the EBF transcription factor Collier (Col) and the HOX factor Antennapedia (Antp), has been proposed to form a niche required to maintain the pool of hematopoietic progenitors (prohemocytes). In contrast with this model, we show here that genetic ablation of the PSC does not cause an increase in blood cell differentiation or a loss of blood cell progenitors. Furthermore, although both col and Antp mutant larvae are devoid of PSC, the massive prohemocyte differentiation observed in col mutant is not phenocopied in Antp mutant. Interestingly, beside its expression in the PSC, Col is also expressed at low levels in prohemocytes and we show that this expression persists in PSC-ablated and Antp mutant larvae. Moreover, targeted knockdown and rescue experiments indicate that Col expression is required in the prohemocytes to prevent their differentiation. Together, our findings show that the PSC is dispensable for blood cell progenitor maintenance and reveal the key role of the conserved transcription factor Col as an intrinsic regulator of hematopoietic progenitor fate.

Pri peptides are mediators of ecdysone for the temporal control of development.

Animal development fundamentally relies on the precise control, in space and time, of genome expression. Whereas we have a wealth of information about spatial patterning, the mechanisms underlying temporal control remain poorly understood. Here we show that Pri peptides, encoded by small open reading frames, are direct mediators of the steroid hormone ecdysone for the timing of developmental programs in Drosophila. We identify a previously uncharacterized enzyme of ecdysone biosynthesis, GstE14, and find that ecdysone triggers pri expression to define the onset of epidermal trichome development, through post-translational control of the Shavenbaby transcription factor. We show that manipulating pri expression is sufficient to either put on hold or induce premature differentiation of trichomes. Furthermore, we find that ecdysone-dependent regulation of pri is not restricted to epidermis and occurs over various tissues and times. Together, these findings provide a molecular framework to explain how systemic hormonal control coordinates specific programs of differentiation with developmental timing.

The Hippo pathway polarizes the actin cytoskeleton during collective migration of Drosophila border cells.

Collective migration of Drosophila border cells depends on a dynamic actin cytoskeleton that is highly polarized such that it concentrates around the outer rim of the migrating cluster of cells. How the actin cytoskeleton becomes polarized in these cells to enable collective movement remains unknown. Here we show that the Hippo signaling pathway links determinants of cell polarity to polarization of the actin cytoskeleton in border cells. Upstream Hippo pathway components localize to contacts between border cells inside the cluster and signal through the Hippo and Warts kinases to polarize actin and promote border cell migration. Phosphorylation of the transcriptional coactivator Yorkie (Yki)/YAP by Warts does not mediate the function of this pathway in promoting border cell migration, but rather provides negative feedback to limit the speed of migration. Instead, Warts phosphorylates and inhibits the actin regulator Ena to activate F-actin Capping protein activity on inner membranes and thereby restricts F-actin polymerization mainly to the outer rim of the migrating cluster.

Dual role for Insulin/TOR signaling in the control of hematopoietic progenitor maintenance in Drosophila.

The interconnected Insulin/IGF signaling (IlS) and Target of Rapamycin (TOR) signaling pathways constitute the main branches of the nutrient-sensing system that couples growth to nutritional conditions in Drosophila. Here, we addressed the influence of these pathways and of diet restriction on the balance between the maintenance of multipotent hematopoietic progenitors and their differentiation in the Drosophila lymph gland. In this larval hematopoietic organ, a pool of stem-like progenitor blood cells (prohemocytes) is kept undifferentiated in response to signaling from a specialized group of cells forming the posterior signaling center (PSC), which serves as a stem cell niche. We show that, reminiscent of the situation in human, loss of the negative regulator of IIS Pten results in lymph gland hyperplasia, aberrant blood cell differentiation and hematopoietic progenitor exhaustion. Using site-directed loss- and gain-of-function analysis, we demonstrate that components of the IIS/TOR pathways control lymph gland homeostasis at two levels. First, they cell-autonomously regulate the size and activity of the hematopoietic niche. Second, they are required within the prohemocytes to control their growth and maintenance. Moreover, we show that diet restriction or genetic alteration mimicking amino acid deprivation triggers progenitor cell differentiation. Hence, our study highlights the role of the IIS/TOR pathways in orchestrating hematopoietic progenitor fate and links blood cell fate to nutritional status.

Modeling cancers in Drosophila.

The basic cellular processes deregulated during carcinogenesis and the vast majority of the genes implicated in cancer appear conserved from humans to flies. This conservation, together with an ever-expanding fly genetic toolbox, has made of Drosophila melanogaster a remarkably profitable model to study many fundamental aspects of carcinogenesis. In particular, Drosophila has played a major role in the identification of genes and pathways implicated in cancer and in disclosing novel functional relationships between cancer genes. It has also proved to be a genetically tractable system where to mimic cancer-like situations and characterize the mode of action of human oncogenes. Here, we outline some advances in the study of cancer, both at the basic and more translational levels, which have benefited from research carried out in flies.

The Hippo pathway regulates intestinal stem cell proliferation during Drosophila adult midgut regeneration.

Intestinal stem cells (ISCs) in the adult Drosophila midgut proliferate to self-renew and to produce differentiating daughter cells that replace those lost as part of normal gut function. Intestinal stress induces the activation of Upd/Jak/Stat signalling, which promotes intestinal regeneration by inducing rapid stem cell proliferation. We have investigated the role of the Hippo (Hpo) pathway in the Drosophila intestine (midgut). Hpo pathway inactivation in either the ISCs or the differentiated enterocytes induces a phenotype similar to that observed under stress situations, including increased stem cell proliferation and expression of Jak/Stat pathway ligands. Hpo pathway targets are induced by stresses such as bacterial infection, suggesting that the Hpo pathway functions as a sensor of cellular stress in the differentiated cells of the midgut. In addition, Yki, the pro-growth transcription factor target of the Hpo pathway, is required in ISCs to drive the proliferative response to stress. Our results suggest that the Hpo pathway is a mediator of the regenerative response in the Drosophila midgut.

LRCH proteins: a novel family of cytoskeletal regulators.

BACKGROUND: Comparative genomics has revealed an unexpected level of conservation for gene products across the evolution of animal species. However, the molecular function of only a few proteins has been investigated experimentally, and the role of many animal proteins still remains unknown. Here we report the characterization of a novel family of evolutionary conserved proteins, which display specific features of cytoskeletal scaffolding proteins, referred to as LRCHs. PRINCIPAL FINDINGS: Taking advantage of the existence of a single LRCH gene in flies, dLRCH, we explored its function in cultured cells, and show that dLRCH act to stabilize the cell cortex during cell division. dLRCH depletion leads to ectopic cortical blebs and alters positioning of the mitotic spindle. We further examined the consequences of dLRCH deletion throughout development and adult life. Although dLRCH is not essential for cell division in vivo, flies lacking dLRCH display a reduced fertility and fitness, particularly when raised at extreme temperatures. CONCLUSION/SIGNIFICANCE: These results support the idea that some cytoskeletal regulators are important to buffer environmental variations and ensure the proper execution of basic cellular processes, such as the control of cell shape, under environmental variations.

An in vivo RNA interference screen identifies gene networks controlling Drosophila melanogaster blood cell homeostasis.

BACKGROUND: In metazoans, the hematopoietic system plays a key role both in normal development and in defense of the organism. In Drosophila, the cellular immune response involves three types of blood cells: plasmatocytes, crystal cells and lamellocytes. This last cell type is barely present in healthy larvae, but its production is strongly induced upon wasp parasitization or in mutant contexts affecting larval blood cell homeostasis. Notably, several zygotic mutations leading to melanotic mass (or "tumor") formation in larvae have been associated to the deregulated differentiation of lamellocytes. To gain further insights into the gene regulatory network and the mechanisms controlling larval blood cell homeostasis, we conducted a tissue-specific loss of function screen using hemocyte-specific Gal4 drivers and UAS-dsRNA transgenic lines. RESULTS: By targeting around 10% of the Drosophila genes, this in vivo RNA interference screen allowed us to recover 59 melanotic tumor suppressor genes. In line with previous studies, we show that melanotic tumor formation is associated with the precocious differentiation of stem-cell like blood progenitors in the larval hematopoietic organ (the lymph gland) and the spurious differentiation of lamellocytes. We also find that melanotic tumor formation can be elicited by defects either in the fat body, the embryo-derived hemocytes or the lymph gland. In addition, we provide a definitive confirmation that lymph gland is not the only source of lamellocytes as embryo-derived plasmatocytes can differentiate into lamellocytes either upon wasp infection or upon loss of function of the Friend of GATA cofactor U-shaped. CONCLUSIONS: In this study, we identify 55 genes whose function had not been linked to blood cell development or function before in Drosophila. Moreover our analyses reveal an unanticipated plasticity of embryo-derived plasmatocytes, thereby shedding new light on blood cell lineage relationship, and pinpoint the Friend of GATA transcription cofactor U-shaped as a key regulator of the plasmatocyte to lamellocyte transformation.

Differential roles of PtdIns(4,5)P2 and phosphorylation in moesin activation during Drosophila development.

The ezrin, radixin and moesin (ERM) proteins regulate cell membrane architecture in several cellular contexts. Current models propose that ERM activation requires a PtdIns(4,5)P(2)-induced conformational change, followed by phosphorylation of a conserved threonine. However, how these inputs contribute in vivo to orchestrate ERM activation is poorly understood. We addressed this issue by evaluating the contribution of PtdIns(4,5)P(2) and phosphorylation to the regulation of moesin during Drosophila development. Unexpectedly, we found that a form of moesin that cannot be phosphorylated displayed significant activity and could substitute for the endogenous product during wing morphogenesis. By contrast, we also show that PtdIns(4,5)P(2) binding is essential for moesin recruitment to the membrane and for its subsequent phosphorylation. Our data indicate that PtdIns(4,5)P(2) acts as a dosing mechanism that locally regulates ERM membrane recruitment and activation, whereas cycles of phosphorylation and dephosphorylation further control their activity once they have reached the cell cortex.

The Hippo pathway regulates apical-domain size independently of its growth-control function.

The Hippo pathway, identified in Drosophila and conserved in vertebrates, regulates tissue growth by promoting cell cycle exit and apoptosis. In addition to their well-characterised overproliferation phenotype, adult Drosophila epithelial cells mutant for the kinases Hippo and Warts have hypertrophic apical domains. Here we examine the molecular basis of this apical hypertrophy and its impact on cell proliferation. In the wing imaginal disc epithelium, we observe increased staining for members of the apical polarity complexes aPKC and Crumbs as well as adherens junction components when Hippo activity is compromised, while basolateral markers are not affected. This increase in apical proteins is correlated with a hypertrophy of the apical domain and adherens junctions. The cell surface localisation of the Notch receptor is also increased in mutant clones, opening the possibility that aberrant receptor signalling may participate in overgrowth of hpo-deficient tissue. Interestingly, however, although the polarity determinant Crumbs is required for the accumulation of apical proteins, this does not appear to significantly contribute to the overproliferation defect elicited by loss of Hippo signalling. Therefore, Hippo signalling controls growth and apical domain size by distinct mechanisms.

Salvador-warts-hippo signaling promotes Drosophila posterior follicle cell maturation downstream of notch.

The Salvador Warts Hippo (SWH) network limits tissue size in Drosophila and vertebrates [1]. Decreased SWH pathway activity gives rise to excess proliferation and reduced apoptosis. The core of the SWH network is composed of two serine/threonine kinases Hippo (Hpo) and Warts (Wts), the scaffold proteins Salvador (Sav) and Mats, and the transcriptional coactivator Yorkie (Yki) [1]. Two band 4.1 related proteins, Merlin (Mer) and Expanded (Ex), have been proposed to act upstream of Hpo, which in turn activates Wts ([1] for review). Wts phosphorylates and inhibits Yki, repressing the expression of Yki target genes [2-4]. Recently, several planar cell polarity (PCP) genes have been implicated in the SWH network in growth control [5-8]. Here, we show that, during oogenesis, the core components of the SWH network are required in posterior follicle cells (PFCs) competent to receive the Gurken (Grk)/TGFalpha signal emitted by the oocyte to control body axis formation. Our results suggest that the SWH network controls the expression of Hindsight, the downstream effector of Notch, required for follicle cell mitotic cycle-endocycle switch. The PCP members of the SWH network are not involved in this process, indicating that signaling upstream of Hpo varies according to developmental context.

The Drosophila RASSF homolog antagonizes the hippo pathway.

Correct organ size is determined by the balance between cell death and proliferation. Perturbation of this delicate balance leads to cancer formation . Hippo (Hpo), the Drosophila ortholog of MST1 and MST2 (Mammalian Sterile 20-like 1 and 2) is a key regulator of a signaling pathway that controls both cell death and proliferation . This pathway is so far composed of two Band 4.1 proteins, Expanded (Ex) and Merlin (Mer), two serine/threonine kinases, Hpo and Warts (Wts), the scaffold proteins Salvador (Sav) and Mats, and the transcriptional coactivator Yorkie (Yki). It has been proposed that Ex and Mer act upstream of Hpo, which in turn phosphorylates and activates Wts. Wts phosphorylates Yki and thus inhibits its activity and reduces expression of Yki target genes such as the caspase inhibitor DIAP1 and the micro RNA bantam. However, the mechanisms leading to Hpo activation are still poorly understood. In mammalian cells, members of the Ras association family (RASSF) of tumor suppressors have been shown to bind to MST1 and modulate its activity . In this study, we show that the Drosophila RASSF ortholog (dRASSF) restricts Hpo activity by competing with Sav for binding to Hpo. In addition, we observe that dRASSF also possesses a tumor-suppressor function.

Dmp53 activates the Hippo pathway to promote cell death in response to DNA damage.

Developmental and environmental signals control a precise program of growth, proliferation, and cell death. This program ensures that animals reach, but do not exceed, their typical size . Understanding how cells sense the limits of tissue size and respond accordingly by exiting the cell cycle or undergoing apoptosis has important implications for both developmental and cancer biology. The Hippo (Hpo) pathway comprises the kinases Hpo and Warts/Lats (Wts), the adaptors Salvador (Sav) and Mob1 as a tumor suppressor (Mats), the cytoskeletal proteins Expanded and Merlin, and the transcriptional cofactor Yorkie (Yki) . This pathway has been shown to restrict cell division and promote apoptosis. The caspase repressor DIAP1 appears to be a primary target of the Hpo pathway in cell-death control. Firstly, Hpo promotes DIAP1 phosphorylation, likely decreasing its stability. Secondly, Wts phosphorylates and inactivates Yki, decreasing DIAP1 transcription. Although we understand some of the events downstream of the Hpo kinase, its mode of activation remains mysterious. Here, we show that Hpo can be activated by Ionizing Radiations (IR) in a Dmp53 (Drosophila melanogaster p53)-dependent manner and that Hpo is required (though not absolutely) for the cell death response elicited by IR or Dmp53 ectopic expression.

Small is beautiful: what flies tell us about ERM protein function in development.

Actin dynamics is recognized as being a determinant in many developmental processes and pathologies, such as cell polarity, morphogenesis and tumour metastasis. However, how actin interacts with the plasma membrane is poorly understood. Although numerous studies in cell culture point to the crucial role of Ezrin, Radixin and Moesin (ERM) proteins in the actin-membrane link, genetic approaches in mice have not yet revealed their activity during development. Drosophila has recently become an alternative and promising system for the genetic study of ERM protein function. This article focuses on advances made in flies, providing evidence for the evolutionary conservation of functional properties of ERM proteins, in addition to shedding new light on their importance for development.

Dmoesin controls actin-based cell shape and polarity during Drosophila melanogaster oogenesis.

Ezrin, Radixin and Moesin (ERM) proteins are thought to constitute a bridge between the actin cytoskeleton and the plasma membrane (PM). Here we report a genetic analysis of Dmoesin, the sole member of the ERM family in Drosophila. We show that Dmoesin is required during oogenesis for anchoring microfilaments to the oocyte cortex. Alteration of the actin cytoskeleton resulting from Dmoesin mutations impairs the localization of maternal determinants, thus disrupting antero-posterior polarity. This study also demonstrates the requirement of Dmoesin for the specific organization of cortical microfilaments in nurse cells and, consequently, mutations in Dmoesin produce severe defects in cell shape.