The Hippo Pathway Regulates Homeostatic Growth of Stem Cell Niche Precursors in the Drosophila Ovary

The Hippo pathway regulates organ size, stem cell proliferation and tumorigenesis in adult organs. Whether the Hippo pathway influences establishment of stem cell niche size to accommodate changes in organ size, however, has received little attention. Here, we ask whether Hippo signaling influences the number of stem cell niches that are established during development of the Drosophila larval ovary, and whether it interacts with the same or different effector signaling pathways in different cell types. We demonstrate that canonical Hippo signaling regulates autonomous proliferation of the soma, while a novel hippo-independent activity of Yorkie regulates autonomous proliferation of the germ line. Moreover, we demonstrate that Hippo signaling mediates non-autonomous proliferation signals between germ cells and somatic cells, and contributes to maintaining the correct proportion of these niche precursors. Finally, we show that the Hippo pathway interacts with different growth pathways in distinct somatic cell types, and interacts with EGFR and JAK/STAT pathways to regulate non-autonomous proliferation of germ cells. We thus provide evidence for novel roles of the Hippo pathway in establishing the precise balance of soma and germ line, the appropriate number of stem cell niches, and ultimately regulating adult female reproductive capacity.     

Dally Proteoglycan Mediates the Autonomous and Nonautonomous Effects on Tissue Growth Caused by Activation of the PI3K and TOR Pathways

How cells acquiring mutations in tumor suppressor genes outcompete neighboring wild-type cells is poorly understood. The phosphatidylinositol 3-kinase (PI3K)–phosphatase with tensin homology (PTEN) and tuberous sclerosis complex (TSC)-target of rapamycin (TOR) pathways are frequently activated in human cancer, and this activation is often causative of tumorigenesis. We utilized the Gal4-UAS system in Drosophila imaginal primordia, highly proliferative and growing tissues, to analyze the impact of restricted activation of these pathways on neighboring wild-type cell populations. Activation of these pathways leads to an autonomous induction of tissue overgrowth and to a remarkable nonautonomous reduction in growth and proliferation rates of adjacent cell populations. This nonautonomous response occurs independently of where these pathways are activated, is functional all throughout development, takes place across compartments, and is distinct from cell competition. The observed autonomous and nonautonomous effects on tissue growth rely on the up-regulation of the proteoglycan Dally, a major element involved in modulating the spreading, stability, and activity of the growth promoting Decapentaplegic (Dpp)/transforming growth factor β(TGF-β) signaling molecule. Our findings indicate that a reduction in the amount of available growth factors contributes to the outcompetition of wild-type cells by overgrowing cell populations. During normal development, the PI3K/PTEN and TSC/TOR pathways play a major role in sensing nutrient availability and modulating the final size of any developing organ. We present evidence that Dally also contributes to integrating nutrient sensing and organ scaling, the fitting of pattern to size.     

Mechanical Control of Organ Size in the Development of the Drosophila Wing Disc

Control of cessation of growth in developing organs has recently been proposed to be influenced by mechanical forces acting on the tissue due to its growth. In particular, it was proposed that stretching of the tissue leads to an increase in cell proliferation. Using the model system of the Drosophila wing imaginal disc, we directly stretch the tissue finding a significant increase in cell proliferation, thus confirming this hypothesis. In addition, we characterize the growth over the entire growth period of the wing disc finding a correlation between the apical cell area and cell proliferation rate.PACS numbers: 87.19.lx, 87.18.Nq, 87.80.Ek, 87.17.Ee, 87.85.Xd     

Cell-autonomous regulation of cell and organ growth in Drosophila by Akt/PKB

Organismal size is determined by a tightly regulated mechanism that coordinates cell growth, cell proliferation and cell death. The Drosophila insulin receptor/Chico/Dp110 pathway regulates cell and organismal size. Here we show that genetic manipulation of the phosphoinositide-3-OH-kinase-dependent serine/threonine protein kinase Akt (protein kinase B) during development of the Drosophila imaginal disc affects cell and organ size in an autonomous manner. Ectopic expression of Akt does not affect cell-fate determination, apoptosis or proliferation rates in imaginal discs. Thus, Akt appears to stimulate intracellular pathways that specifically regulate cell and compartment size independently of cell proliferation in vivo.     

E2F and p53 induce apoptosis independently during Drosophila development but intersect in the context of DNA damage

In mammalian cells, RB/E2F and p53 are intimately connected, and crosstalk between these pathways is critical for the induction of cell cycle arrest or cell death in response to cellular stresses. Here we have investigated the genetic interactions between RBF/E2F and p53 pathways during Drosophila development. Unexpectedly, we find that the pro-apoptotic activities of E2F and p53 are independent of one another when examined in the context of Drosophila development: apoptosis induced by the deregulation of dE2F1, or by the overexpression of dE2F1, is unaffected by the elimination of dp53; conversely, dp53-induced phenotypes are unaffected by the elimination of dE2F activity. However, dE2F and dp53 converge in the context of a DNA damage response. Both dE2F1/dDP and dp53 are required for DNA damage-induced cell death, and the analysis of rbf1 mutant eye discs indicates that dE2F1/dDP and dp53 cooperatively promote cell death in irradiated discs. In this context, the further deregulation in the expression of pro-apoptotic genes generates an additional sensitivity to apoptosis that requires both dE2F/dDP and dp53 activity. This sensitivity differs from DNA damage-induced apoptosis in wild-type discs (and from dE2F/dDP-induced apoptosis in un-irradiated rbf1 mutant eye discs) by being dependent on both hid and reaper. These results show that pro-apoptotic activities of dE2F1 and dp53 are surprisingly separable: dp53 is required for dE2F-dependent apoptosis in the response to DNA damage, but it is not required for dE2F-dependent apoptosis caused simply by the inactivation of rbf1.     

Altered modes of stem cell division drive adaptive intestinal growth

Throughout life, adult organs continually adapt to variable environmental factors. Adaptive mechanisms must fundamentally differ from homeostatic maintenance, but little is known about how physiological factors elicit tissue remodeling. Here, we show that specialized stem cell responses underlie the adaptive resizing of a mature organ. In the adult Drosophila midgut, intestinal stem cells interpret a nutrient cue to "break homeostasis" and drive growth when food is abundant. Activated in part by niche production of insulin, stem cells direct a growth program through two altered modes of behavior: accelerated division rates and predominance of symmetric division fates. Together, these altered modes produce a net increase in total intestinal cells, which is reversed upon withdrawal of food. Thus, tissue renewal programs are not committed to maintain cellular equilibrium; stem cells can remodel organs in response to physiological triggers.     

Shar-pei mediates cell proliferation arrest during imaginal disc growth in Drosophila

During animal development, organ size is determined primarily by the amount of cell proliferation, which must be tightly regulated to ensure the generation of properly proportioned organs. However, little is known about the molecular pathways that direct cells to stop proliferating when an organ has attained its proper size. We have identified mutations in a novel gene, shar-pei, that is required for proper termination of cell proliferation during Drosophila imaginal disc development. Clones of shar-pei mutant cells in imaginal discs produce enlarged tissues containing more cells of normal size. We show that this phenotype is the result of both increased cell proliferation and reduced apoptosis. Hence, shar-pei restricts cell proliferation and promotes apoptosis. By contrast, shar-pei is not required for cell differentiation and pattern formation of adult tissue. Shar-pei is also not required for cell cycle exit during terminal differentiation, indicating that the mechanisms directing cell proliferation arrest during organ growth are distinct from those directing cell cycle exit during terminal differentiation. shar-pei encodes a WW-domain-containing protein that has homologs in worms, mice and humans, suggesting that mechanisms of organ growth control are evolutionarily conserved.     

A dp53/JNK-dependant feedback amplification loop is essential for the apoptotic response to stress in Drosophila

Programmed cell death (apoptosis) is a conserved process aimed to eliminate unwanted cells. The key molecules are a group of proteases called caspases that cleave vital proteins, which leads to the death of cells. In Drosophila, the apoptotic pathway is usually represented as a cascade of events in which an initial stimulus activates one or more of the proapoptotic genes (hid, rpr, grim), which in turn activate caspases. In stress-induced apoptosis, the dp53 (Drosophila p53) gene and the Jun N-terminal kinase (JNK) pathway function upstream in the activation of the proapoptotic genes. Here we demonstrate that dp53 and JNK also function downstream of proapoptotic genes and the initiator caspase Dronc (Drosophila NEDD2-like caspase) and that they establish a feedback loop that amplifies the initial apoptotic stimulus. This loop plays a critical role in the apoptotic response because in its absence there is a dramatic decrease in the amount of cell death after a pulse of the proapoptotic proteins Hid and Rpr. Thus, our results indicate that stress-induced apoptosis in Drosophila is dependant on an amplification loop mediated by dp53 and JNK. Furthermore, they also demonstrate a mechanism of mutual activation of proapoptotic genes.     

Tuberous sclerosis complex and Myc coordinate the growth and division of Drosophila intestinal stem cells

Intestinal stem cells (ISCs) in the adult Drosophila melanogaster midgut can respond to damage and support repair. We demonstrate in this paper that the tuberous sclerosis complex (TSC) plays a critical role in balancing ISC growth and division. Previous studies have shown that imaginal disc cells that are mutant for TSC have increased rates of growth and division. However, we report in this paper that loss of TSC in the adult Drosophila midgut results in the formation of much larger ISCs that have halted cell division. These mutant ISCs expressed proper stem cell markers, did not differentiate, and had defects in multiple steps of the cell cycle. Slowing the growth by feeding rapamycin or reducing Myc was sufficient to rescue the division defect. The TSC mutant guts had a thinner epithelial structure than wild-type tissues, and the mutant flies were more susceptible to tissue damage. Therefore, we have uncovered a context-dependent phenotype of TSC mutants in adult ISCs, such that the excessive growth leads to inhibition of division.     

Nitric oxide and Drosophila development

Mechanisms controlling the transition of precursor cells from proliferation to differentiation during organism development determine the distinct anatomical features of tissues and organs. NO may mediate such a transition since it can suppress DNA synthesis and cell proliferation. Inhibition of NOS activity in the imaginal discs of Drosophila larvae results in hypertrophy of tissues and organs of the adult fly, whereas ectopic overexpression of NOS has the reciprocal, hypotrophic, effect. Furthermore, NO production is crucial for the establishment of ordered neuronal connections in the visual system of the fly, indicating that NO affects the acquisition of the differentiated phenotype by the neural tissue. Increasing evidence points to a broad role that NO may play in animal development by acting as an essential negative regulator of precursor cell proliferation during tissue and organ morphogenesis.     

Induction of apoptosis by Drosophila Myc

Myc proteins are essential regulators of cellular growth and proliferation during normal development. Activating mutations in myc genes result in excessive growth and are frequently associated with human cancers. At the same time, forced expression of Myc sensitizes vertebrate cells towards different pro-apoptotic stimuli. Recently, the ability of overexpressed Myc to induce cell-autonomous apoptosis has been shown to be evolutionarily conserved in Drosophila Myc (dMyc). Here, we show that dMyc induced apoptosis is accompanied by the induction of Drosophila p53 mRNA, but that dp53 activity is not essential for dMyc's ability to induce apoptosis. Conversely, larvae carrying a hypomorphic dmyc mutation are more resistant to the apoptosis-promoting effects of X-irradiation. These data suggest that the control of apoptosis is a physiological function of Myc and that dMyc might play a role in the response to DNA damage.     

Morphogen control of wing growth through the Fat signaling pathway

Organ growth is influenced by organ patterning, but the molecular mechanisms that link patterning to growth have remained unclear. We show that the Dpp morphogen gradient in the Drosophila wing influences growth by modulating the activity of the Fat signaling pathway. Dpp signaling regulates the expression and localization of Fat pathway components, and Fat signaling through Dachs is required for the effect of the Dpp gradient on cell proliferation. Juxtaposition of cells that express different levels of the Fat pathway regulators four-jointed and dachsous stimulates expression of Fat/Hippo pathway target genes and cell proliferation, consistent with the hypothesis that the graded expression of these genes contributes to wing growth. Moreover, uniform expression of four-jointed and dachsous in the wing inhibits cell proliferation. These observations identify Fat as a signaling pathway that links the morphogen-mediated establishment of gradients of positional values across developing organs to the regulation of organ growth.     

Notch-mediated repression of bantam miRNA contributes to boundary formation in the Drosophila wing

Subdivision of proliferating tissues into adjacent compartments that do not mix plays a key role in animal development. The Actin cytoskeleton has recently been shown to mediate cell sorting at compartment boundaries, and reduced cell proliferation in boundary cells has been proposed as a way of stabilizing compartment boundaries. Cell interactions mediated by the receptor Notch have been implicated in the specification of compartment boundaries in vertebrates and in Drosophila, but the molecular effectors remain largely unidentified. Here, we present evidence that Notch mediates boundary formation in the Drosophila wing in part through repression of bantam miRNA. bantam induces cell proliferation and we have identified the Actin regulator Enabled as a new target of bantam. Increased levels of Enabled and reduced proliferation rates contribute to the maintenance of the dorsal-ventral affinity boundary. The activity of Notch also defines, through the homeobox-containing gene cut, a distinct population of boundary cells at the dorsal-ventral (DV) interface that helps to segregate boundary from non-boundary cells and contributes to the maintenance of the DV affinity boundary.     

Lgl/aPKC and Crb regulate the Salvador/Warts/Hippo pathway

A key goal of developmental biology is to understand the mechanisms that coordinate organ growth. It has long been recognized that the genes that control apico-basal cell polarity also regulate tissue growth. How loss of cell polarity contributes to tissue overgrowth has been the subject of much speculation. Do loss-of-function mutations in cell polarity regulators result in secondary effects that globally deregulate cell proliferation, or do these genes specifically control growth pathways? Three recent papers have shown that the apico-basal polarity determinants Lgl/aPKC and Crb regulate tissue growth independently of their roles in cell polarity and coordinately regulate cell proliferation and cell death via the Salvador/Warts/Hippo (SWH) pathway. Lgl/aPKC are required for the correct localization of Hippo (Hpo)/Ras associated factor (RASSF), while Crb regulates the levels and localization of Expanded (Ex), indicating that cell polarity determinants modify SWH pathway activity by distinct mechanisms. Here, we review the key data that support these conclusions, highlight remaining questions and speculate on the underlying mechanisms by which the cell polarity complexes interact with the SWH pathway. Understanding the interactions between cell polarity regulators and the SWH pathway will improve our knowledge of how epithelial organization and tissue growth are coordinated during development and perturbed in disease states such as cancer.Key words: Drosophila, tumor suppressor gene, cell polarity, Hippo pathway, Crb, Lgl, aPKC     

Identification and characterization of genes required for compensatory growth in Drosophila

To maintain tissue homeostasis, some organs are able to replace dying cells with additional proliferation of surviving cells. Such proliferation can be localized (e.g., a regeneration blastema) or diffuse (compensatory growth). The relationship between such growth and the growth that occurs during development has not been characterized in detail. Drosophila melanogaster larval imaginal discs can recover from extensive damage, producing normally sized adult organs. Here we describe a system using genetic mosaics to screen for recessive mutations that impair compensatory growth. By generating clones of cells that carry a temperature-sensitive cell-lethal mutation, we conditionally ablate patches of tissue in the imaginal disc and assess the ability of the surviving sister clones to replace the lost tissue. We have used this system together with a modified whole-genome resequencing (WGS) strategy to identify several mutations that selectively compromise compensatory growth. We find specific alleles of bunched (bun) and Ribonucleoside diphosphate reductase large subunit (RnrL) reduce compensatory growth in the imaginal disc. Other genes identified in the screen, including two alleles of Topoisomerase 3-alpha (Top3α), while also required for developmental growth, appear to have an enhanced requirement during compensatory growth. Compensatory growth occurs at a higher rate than normal growth and may therefore have features in common with some types of overgrowth. Indeed, the RnrL allele identified compromises both these types of altered growth and mammalian ribonucleotide reductase and topoisomerases are targets of anticancer drugs. Finally, the approach we describe is applicable to the study of compensatory growth in diverse tissues in Drosophila.