Elimination of aberrantly specified cell clones is independent of interfacial Myosin II accumulation.

Spatial organization within an organ is essential and needs to be maintained during development. This is largely implemented via compartment boundaries that serve as barriers between distinct cell types. Biased accumulation of junctional non-muscle Myosin II along the interface between differently fated groups of cells contributes to boundary integrity and maintains its shape via increased tension. Here, using the Drosophila wing imaginal disc, we tested whether interfacial tension driven by accumulation of Myosin is responsible for the elimination of aberrantly specified cells that would otherwise compromise compartment organization. To this end, we genetically reduced Myosin II levels in three different patterns: in both wild-type and misspecified cells, only in misspecified cells, and specifically at the interface between wild-type and aberrantly specified cells. We found that the recognition and elimination of aberrantly specified cells do not strictly rely on tensile forces driven by interfacial Myosin cables. Moreover, apical constriction of misspecified cells and their separation from wild-type neighbours occurred even when Myosin levels were greatly reduced. Thus, we conclude that the forces that drive elimination of aberrantly specified cells are largely independent of Myosin II accumulation.

Correction: Ferritin heavy chain protects the developing wing from reactive oxygen species and ferroptosis.

[This corrects the article DOI: 10.1371/journal.pgen.1008396.].

Cell elimination strategies upon identity switch via modulation of apterous in Drosophila wing disc.

The ability to establish spatial organization is an essential feature of any developing tissue and is achieved through well-defined rules of cell-cell communication. Maintenance of this organization requires elimination of cells with inappropriate positional identity, a poorly understood phenomenon. Here we studied mechanisms regulating cell elimination in the context of a growing tissue, the Drosophila wing disc and its dorsal determinant Apterous. Systematic analysis of apterous mutant clones along with their twin spots shows that they are eliminated from the dorsal compartment via three different mechanisms: relocation to the ventral compartment, basal extrusion, and death, depending on the position of the clone in the wing disc. We find that basal extrusion is the main elimination mechanism in the hinge, whereas apoptosis dominates in the pouch and in the notum. In the absence of apoptosis, extrusion takes over to ensure clearance in all regions. Notably, clones in the hinge grow larger than those in the pouch, emphasizing spatial differences. Mechanistically, we find that limiting cell division within the clones does not prevent their extrusion. Indeed, even clones of one or two cells can be extruded basally, demonstrating that the clone size is not the main determinant of the elimination mechanism to be used. Overall, we revealed three elimination mechanisms and their spatial biases for preserving pattern in a growing organ.

Ferritin heavy chain protects the developing wing from reactive oxygen species and ferroptosis.

The interplay between signalling pathways and metabolism is crucial for tissue growth. Yet, it remains poorly understood. Here, we studied the consequences of modulating iron metabolism on the growth of Drosophila imaginal discs. We find that reducing the levels of the ferritin heavy chain in the larval wing discs leads to drastic growth defects, whereas light chain depletion causes only minor defects. Mutant cell clones for the heavy chain lack the ability to compete against Minute mutant cells. Reactive oxygen species (ROS) accumulate in wing discs with reduced heavy chain levels, causing severe mitochondrial defects and ferroptosis. Preventing ROS accumulation alleviates some of the growth defects. We propose that the increased expression of ferritin in hippo mutant cells may protect against ROS accumulation.

Dpp spreading is required for medial but not for lateral wing disc growth.

Drosophila Decapentaplegic (Dpp) has served as a paradigm to study morphogen-dependent growth control. However, the role of a Dpp gradient in tissue growth remains highly controversial. Two fundamentally different models have been proposed: the 'temporal rule' model suggests that all cells of the wing imaginal disc divide upon a 50% increase in Dpp signalling, whereas the 'growth equalization model' suggests that Dpp is only essential for proliferation control of the central cells. Here, to discriminate between these two models, we generated and used morphotrap, a membrane-tethered anti-green fluorescent protein (GFP) nanobody, which enables immobilization of enhanced (e)GFP::Dpp on the cell surface, thereby abolishing Dpp gradient formation. We find that in the absence of Dpp spreading, wing disc patterning is lost; however, lateral cells still divide at normal rates. These data are consistent with the growth equalization model, but do not fit a global temporal rule model in the wing imaginal disc.

Rounding up the Usual Suspects: Assessing Yorkie, AP-1, and Stat Coactivation in Tumorigenesis.

Can hyperactivation of a few key signaling effectors be the underlying reason for the majority of epithelial cancers despite different driver mutations? Here, to address this question, we use the Drosophila model, which allows analysis of gene expression from tumors with known initiating mutations. Furthermore, its simplified signaling pathways have numerous well characterized targets we can use as pathway readouts. In Drosophila tumor models, changes in the activities of three pathways, Jun N-terminal Kinase (JNK), Janus Kinase / Signal Transducer and Activator of Transcription (JAK/STAT), and Hippo, mediated by AP-1 factors, Stat92E, and Yorkie, are reported frequently. We hypothesized this may indicate that these three pathways are commonly deregulated in tumors. To assess this, we mined the available transcriptomic data and evaluated the activity levels of eight pathways in various tumor models. Indeed, at least two out of our three suspects contribute to tumor development in all Drosophila cancer models assessed, despite different initiating mutations or tissues of origin. Surprisingly, we found that Notch signaling is also globally activated in all models examined. We propose that these four pathways, JNK, JAK/STAT, Hippo, and Notch, are paid special attention and assayed for systematically in existing and newly developed models.

Establishment of a Developmental Compartment Requires Interactions between Three Synergistic Cis-regulatory Modules.

The subdivision of cell populations in compartments is a key event during animal development. In Drosophila, the gene apterous (ap) divides the wing imaginal disc in dorsal vs ventral cell lineages and is required for wing formation. ap function as a dorsal selector gene has been extensively studied. However, the regulation of its expression during wing development is poorly understood. In this study, we analyzed ap transcriptional regulation at the endogenous locus and identified three cis-regulatory modules (CRMs) essential for wing development. Only when the three CRMs are combined, robust ap expression is obtained. In addition, we genetically and molecularly analyzed the trans-factors that regulate these CRMs. Our results propose a three-step mechanism for the cell lineage compartment expression of ap that includes initial activation, positive autoregulation and Trithorax-mediated maintenance through separable CRMs.

Differential regulation of the Hippo pathway by adherens junctions and apical-basal cell polarity modules.

Adherens junctions (AJs) and cell polarity complexes are key players in the establishment and maintenance of apical-basal cell polarity. Loss of AJs or basolateral polarity components promotes tumor formation and metastasis. Recent studies in vertebrate models show that loss of AJs or loss of the basolateral component Scribble (Scrib) cause deregulation of the Hippo tumor suppressor pathway and hyperactivation of its downstream effectors Yes-associated protein (YAP) and Transcriptional coactivator with PDZ-binding motif (TAZ). However, whether AJs and Scrib act through the same or independent mechanisms to regulate Hippo pathway activity is not known. Here, we dissect how disruption of AJs or loss of basolateral components affect the activity of the Drosophila YAP homolog Yorkie (Yki) during imaginal disc development. Surprisingly, disruption of AJs and loss of basolateral proteins produced very different effects on Yki activity. Yki activity was cell-autonomously decreased but non-cell-autonomously elevated in tissues where the AJ components E-cadherin (E-cad) or α-catenin (α-cat) were knocked down. In contrast, scrib knockdown caused a predominantly cell-autonomous activation of Yki. Moreover, disruption of AJs or basolateral proteins had different effects on cell polarity and tissue size. Simultaneous knockdown of α-cat and scrib induced both cell-autonomous and non-cell-autonomous Yki activity. In mammalian cells, knockdown of E-cad or α-cat caused nuclear accumulation and activation of YAP without overt effects on Scrib localization and vice versa. Therefore, our results indicate the existence of multiple, genetically separable inputs from AJs and cell polarity complexes into Yki/YAP regulation.

Dpp/BMP signaling in flies: from molecules to biology.

Decapentaplegic (Dpp), the fly homolog of the secreted mammalian BMP2/4 signaling molecules, is involved in almost all aspects of fly development. Dpp has critical functions at all developmental stages, from patterning of the eggshell to the determination of adult intestinal stem cell identity. Here, we focus on recent findings regarding the transcriptional regulatory logic of the pathway, on a new feedback regulator, Pentagone, and on Dpp's roles in scaling and growth of the Drosophila wing.

Dpp signaling activity requires Pentagone to scale with tissue size in the growing Drosophila wing imaginal disc.

The wing of the fruit fly, Drosophila melanogaster, with its simple, two-dimensional structure, is a model organ well suited for a systems biology approach. The wing arises from an epithelial sac referred to as the wing imaginal disc, which undergoes a phase of massive growth and concomitant patterning during larval stages. The Decapentaplegic (Dpp) morphogen plays a central role in wing formation with its ability to co-coordinately regulate patterning and growth. Here, we asked whether the Dpp signaling activity scales, i.e. expands proportionally, with the growing wing imaginal disc. Using new methods for spatial and temporal quantification of Dpp activity and its scaling properties, we found that the Dpp response scales with the size of the growing tissue. Notably, scaling is not perfect at all positions in the field and the scaling of target gene domains is ensured specifically where they define vein positions. We also found that the target gene domains are not defined at constant concentration thresholds of the downstream Dpp activity gradients P-Mad and Brinker. Most interestingly, Pentagone, an important secreted feedback regulator of the pathway, plays a central role in scaling and acts as an expander of the Dpp gradient during disc growth.

The tumour-suppressor genes NF2/Merlin and Expanded act through Hippo signalling to regulate cell proliferation and apoptosis.

Merlin, the protein product of the Neurofibromatosis type-2 gene, acts as a tumour suppressor in mice and humans. Merlin is an adaptor protein with a FERM domain and it is thought to transduce a growth-regulatory signal. However, the pathway through which Merlin acts as a tumour suppressor is poorly understood. Merlin, and its function as a negative regulator of growth, is conserved in Drosophila, where it functions with Expanded, a related FERM domain protein. Here, we show that Drosophila Merlin and Expanded are components of the Hippo signalling pathway, an emerging tumour-suppressor pathway. We find that Merlin and Expanded, similar to other components of the Hippo pathway, are required for proliferation arrest and apoptosis in developing imaginal discs. Our genetic and biochemical data place Merlin and Expanded upstream of Hippo and identify a pathway through which they act as tumour-suppressor genes.

The apical-basal cell polarity determinant Crumbs regulates Hippo signaling in Drosophila.

Defects in apical-basal cell polarity and abnormal expression of cell polarity determinants are often associated with cancer in vertebrates. In Drosophila, abnormal expression of apical-basal determinants can cause neoplastic phenotypes, including loss of cell polarity and overproliferation. However, the pathways through which apical-basal polarity determinants affect growth are poorly understood. Here, we investigated the mechanism by which the apical determinant Crumbs (Crb) affects growth in Drosophila imaginal discs. Overexpression of Crb causes severe overproliferation, and we found that loss of Crb similarly results in overgrowth of imaginal discs. Crb gain and loss of function caused defects in Hippo signaling, a key signaling pathway that controls tissue growth in Drosophila and mammals. Manipulation of Crb levels caused the up-regulation of Hippo target genes, genetically interacted with known Hippo pathway components, and required Yorkie, a transcriptional coactivator that acts downstream in the Hippo pathway, for target gene induction and overgrowth. Interestingly, Crb regulates growth and cell polarity through different motifs in its intracellular domain. A juxtamembrane FERM domain-binding motif is responsible for growth regulation and induction of Hippo target gene expression, whereas Crb uses a PDZ-binding motif to form a complex with other polarity factors. The Hippo pathway component Expanded, an apically localized adaptor protein, is mislocalized in both crb mutant cells and Crb overexpressing tissues, whereas the other Hippo pathway components, Fat and Merlin, are unaffected. Taken together, our data show that Crb regulates growth through Hippo signaling, and thus identify Crb as a previously undescribed upstream input into the Hippo pathway.

Boundaries of Dachsous Cadherin activity modulate the Hippo signaling pathway to induce cell proliferation.

The conserved Hippo tumor suppressor pathway is a key signaling pathway that controls organ size in Drosophila. To date a signal transduction cascade from the Cadherin Fat at the plasma membrane into the nucleus has been discovered. However, how the Hippo pathway is regulated by extracellular signals is poorly understood. Fat not only regulates growth but also planar cell polarity, for which it interacts with the Dachsous (Ds) Cadherin, and Four-jointed (Fj), a transmembrane kinase that modulates the interaction between Ds and Fat. Ds and Fj are expressed in gradients and manipulation of their expression causes abnormal growth. However, how Ds and Fj regulate growth and whether they act through the Hippo pathway is not known. Here, we report that Ds and Fj regulate Hippo signaling to control growth. Interestingly, we found that Ds/Fj regulate the Hippo pathway through a remarkable logic. Induction of Hippo target genes is not proportional to the amount of Ds or Fj presented to a cell, as would be expected if Ds and Fj acted as traditional ligands. Rather, Hippo target genes are up-regulated when neighboring cells express different amounts of Ds or Fj. Consistent with a model that differences in Ds/Fj levels between cells regulate the Hippo pathway, we found that artificial Ds/Fj boundaries induce extra cell proliferation, whereas flattening the endogenous Ds and Fj gradients results in growth defects. The Ds/Fj signaling system thus defines a cell-to-cell signaling mechanism that regulates the Hippo pathway, thereby contributing to the control of organ size.

The fat cadherin acts through the hippo tumor-suppressor pathway to regulate tissue size.

BACKGROUND: The Hippo tumor-suppressor pathway has emerged as a key signaling pathway that controls tissue size in Drosophila. Merlin, the Drosophila homolog of the human Neurofibromatosis type-2 (NF2) tumor-suppressor gene, and the related protein Expanded are the most upstream components of the Hippo pathway identified so far. However, components acting upstream of Expanded and Merlin, such as transmembrane receptors, have not yet been identified. RESULTS: Here, we report that the protocadherin Fat acts as an upstream component in the Hippo pathway. Fat is a known tumor-suppressor gene in Drosophila, and fat mutants have severely overgrown imaginal discs. We found that the overgrowth phenotypes of fat mutants are similar to those of mutants in Hippo pathway components: fat mutant cells continued to proliferate after wild-type cells stopped proliferating, and fat mutant cells deregulated Hippo target genes such as cyclin E and diap1. Fat acts genetically and biochemically upstream of other Hippo pathway components such as Expanded, the Hippo and Warts kinases, and the transcriptional coactivator Yorkie. Fat is required for the stability of Expanded and its localization to the plasma membrane. In contrast, Fat is not required for Merlin localization, and Fat and Merlin act in parallel in growth regulation. CONCLUSIONS: Taken together, our data identify a cell-surface molecule that may act as a receptor of the Hippo signaling pathway.

Hippo Reprograms the Transcriptional Response to Ras Signaling.

Hyperactivating mutations in Ras signaling are hallmarks of carcinomas. Ras signaling mediates cell fate decisions as well as proliferation during development. It is not known what dictates whether Ras signaling drives differentiation versus proliferation. Here we show that the Hippo pathway is critical for this decision. Loss of Hippo switches Ras activation from promoting cellular differentiation to aggressive cellular proliferation. Transcriptome analysis combined with genetic tests show that this excessive proliferation depends on the synergistic induction of Ras target genes. Using ChIP-nexus, we find that Hippo signaling keeps Ras targets in check by directly regulating the expression of two key downstream transcription factors of Ras signaling: the ETS-domain transcription factor Pointed and the repressor Capicua. Our results highlight how independent signaling pathways can impinge on each other at the level of transcription factors, thereby providing a safety mechanism to keep proliferation in check under normal developmental conditions.

An Ectopic Network of Transcription Factors Regulated by Hippo Signaling Drives Growth and Invasion of a Malignant Tumor Model.

Cancer cells have abnormal gene expression profiles; however, to what degree these are chaotic or driven by structured gene regulatory networks is often not known. Here we studied a model of Ras-driven invasive tumorigenesis in Drosophila epithelial tissues and combined in vivo genetics with next-generation sequencing and computational modeling to decipher the regulatory logic of tumor cells. Surprisingly, we discovered that the bulk of the tumor-specific gene expression is controlled by an ectopic network of a few transcription factors that are overexpressed and/or hyperactivated in tumor cells. These factors are Stat, AP-1, the bHLH proteins Myc and AP-4, the nuclear hormone receptor Ftz-f1, the nuclear receptor coactivator Taiman/SRC3, and Mef2. Notably, many of these transcription factors also are hyperactivated in human tumors. Bioinformatic analysis predicted that these factors directly regulate the majority of the tumor-specific gene expression, that they are interconnected by extensive cross-regulation, and that they show a high degree of co-regulation of target genes. Indeed, the factors of this network were required in multiple epithelia for tumor growth and invasiveness, and knockdown of several factors caused a reversion of the tumor-specific expression profile but had no observable effect on normal tissues. We further found that the Hippo pathway effector Yorkie was strongly activated in tumor cells and initiated cellular reprogramming by activating several transcription factors of this network. Thus, modeling regulatory networks identified an ectopic and ordered network of master regulators that control a large part of tumor cell-specific gene expression.

Confronting morphogen gradients: how important are they for growth?

Morphogens are crucial for regulating the patterning and the growth of organs. In the developing fly wing, graded distributions of the morphogens Decapentaplegic and Wingless are essential for tissue patterning, but when it comes to growth, evidence suggests that it may not be so.

The Hippo tumor-suppressor pathway regulates apical-domain size in parallel to tissue growth.

The Hippo tumor-suppressor pathway controls tissue growth in Drosophila and mammals by regulating cell proliferation and apoptosis. The Hippo pathway includes the Fat cadherin, a transmembrane protein, which acts upstream of several other components that form a kinase cascade that culminates in the regulation of gene expression through the transcriptional coactivator Yorkie (Yki). Our previous work in Drosophila indicated that Merlin (Mer) and Expanded (Ex) are members of the Hippo pathway and act upstream of the Hippo kinase. In contrast to this model, it was suggested that Mer and Ex primarily regulate membrane dynamics and receptor trafficking, thereby affecting Hippo pathway activity only indirectly. Here, we examined the effects of Mer, Ex and the Hippo pathway on the size of the apical membrane and on apical-basal polarity complexes. We found that mer;ex double mutant imaginal disc cells have significantly increased levels of apical membrane determinants, such as Crb, aPKC and Patj. These phenotypes were shared with mutations in other Hippo pathway components and required Yki, indicating that Mer and Ex signal through the Hippo pathway. Interestingly, however, whereas Crb was required for the accumulation of other apical proteins and for the expansion of the apical domain observed in Hippo pathway mutants, its elimination did not significantly reverse the overgrowth phenotype of warts mutant cells. Therefore, Hippo signaling regulates cell polarity complexes in addition to and independently of its growth control function in imaginal disc cells.

Cryopreservation of starfish oocytes.

Research from many laboratories over the past several decades indicates that invertebrate oocytes and eggs are extraordinarily difficult to freeze. Since starfish oocytes, eggs, and embryos are an important cell and developmental biology model system, there is great interest to cryopreserve these cells. Previous starfish oocyte cryopreservation studies using slow cooling protocols revealed that these cells are highly sensitive to osmotic stress and form intracellular ice at very high sub-zero temperatures, suggesting that common freezing methodologies may not prove useful. We report here that a short exposure to 1.5 M Me2SO/1 M trehalose in hypotonic salt solution followed by ultra-rapid cooling to cryogenic temperatures allows starfish oocytes to be cryopreserved with the average survival rate of 34% when normalized to control oocytes that were exposed to CPA, but not frozen. On average, 51% of the oocytes in 77% of the batches of frozen oocytes underwent meiotic maturation in response to the starfish maturation hormone, 1-methyladenine. In one experiment, eggs developing from thawed oocytes were capable of being fertilized and two developed into embryos. These data suggests that successful cryopreservation of starfish oocytes is possible, but will need further refinement to increase the numbers of fully competent embryos.

MAP kinases regulate unfertilized egg apoptosis and fertilization suppresses death via Ca2+ signaling.

The default fate for eggs from many species is death by apoptosis and thus, successful fertilization depends upon suppression of the maternal death program. Little is known about the molecular triggers which activate this process or how the fertilization signal suppresses the default maternal apoptotic pathway. The MAP kinase (MAPK) family member, ERK, plays a universal and critical role in several stages of oocyte meiotic maturation, and fertilization results in ERK inactivation. In somatic cells, ERK and other MAPK family members, p38 and JNK, provide opposing signals to regulate apoptosis, however, it is not known whether MAPKs play a regulatory role in egg apoptosis, nor whether suppression of apoptosis by fertilization is mediated by MAPK activity. Here we demonstrate that MAPKs are involved in starfish egg apoptosis and we investigate the relationship between the fertilization induced signaling pathway and MAPK activation. ERK is active in post-meiotic eggs just until apoptosis onset and then p38, JNK and a third kinase are activated, and remain active through execution. Sequential activation of ERK and p38 is necessary for apoptosis, and newly synthesized proteins are required both upstream of ERK and downstream of p38 for activation of the full apoptotic program. Fertilization causes a dramatic rise in intracellular Ca2+, and we report that Ca2+ provides a necessary and sufficient pro-survival signal. The Ca2+ pathway following fertilization of both young and aged eggs causes ERK to be rapidly inactivated, but fertilization cannot rescue aged eggs from death, indicating that ERK inactivation is not sufficient to suppress apoptosis.