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Oriented cell division is a fundamental mechanism to control asymmetric stem cell division, neural tube elongation and body axis extension, among other processes. During zebrafish gastrulation, when the body axis extends, dorsal epiblast cells display divisions that are robustly oriented along the animal-vegetal embryonic axis. Here, we use a combination of lipidomics, metabolic tracer analysis and quantitative image analysis to show that sphingolipids mediate spindle positioning during oriented division of epiblast cells. We identify the Wnt signaling as a regulator of sphingolipid synthesis that mediates the activity of serine palmitoyltransferase (SPT), the first and rate-limiting enzyme in sphingolipid production. Sphingolipids determine the palmitoylation state of the Anthrax receptor, which then positions the mitotic spindle of dividing epiblast cells. Our data show how Wnt signaling mediates sphingolipid-dependent oriented division and how sphingolipids determine Anthrax receptor palmitoylation, which ultimately controls the activation of Diaphanous to mediate spindle rotation and oriented mitosis., During development, oriented cell division is important to proper body axis extension. Here, the authors show that sphingolipids are required to direct spindle rotation and oriented mitosis via Anthrax receptor palmitoylation in zebrafish gastrulation.

Notch pathway signaling is implicated in several human cancers. Aberrant activation and mutations of Notch signaling components are linked to tumor initiation, maintenance, and resistance to cancer therapy. Several strategies, such as monoclonal antibodies against Notch ligands and receptors, as well as small-molecule gamma-secretase inhibitors (GSIs), have been developed to interfere with Notch receptor activation at proximal points in the pathway. However, the use of drug-like small molecules to target the down-stream mediators of Notch signaling, the Notch transcription acti-vation complex, remains largely unexplored. Here, we report the discovery of an orally active small-molecule inhibitor (termed CB -103) of the Notch transcription activation complex. We show that CB-103 inhibits Notch signaling in primary human T cell acute lym-phoblastic leukemia and other Notch-dependent human tumor cell lines, and concomitantly induces cell cycle arrest and apoptosis, thereby impairing proliferation, including in GSI-resistant human tumor cell lines with chromosomal translocations and rearrange-ments in Notch genes. CB-103 produces Notch loss-of-function phenotypes in flies and mice and inhibits the growth of human breast cancer and leukemia xenografts, notably without causing the dose-limiting intestinal toxicity associated with other Notch inhibitors. Thus, we describe a pharmacological strategy that in-terferes with Notch signaling by disrupting the Notch transcription complex and shows therapeutic potential for treating Notch -driven cancers.

Cells and organelles are delimited by lipid bilayers in which high deformability is essential to many cell processes, including motility, endocytosis and cell division. Membrane tension is therefore a major regulator of the cell processes that remodel membranes, albeit one that is very hard to measure in vivo. Here we show that a planarizable push-pull fluorescent probe called FliptR (fluorescent lipid tension reporter) can monitor changes in membrane tension by changing its fluorescence lifetime as a function of the twist between its fluorescent groups. The fluorescence lifetime depends linearly on membrane tension within cells, enabling an easy quantification of membrane tension by fluorescence lifetime imaging microscopy. We further show, using model membranes, that this linear dependency between lifetime of the probe and membrane tension relies on a membrane-tension-dependent lipid phase separation. We also provide calibration curves that enable accurate measurement of membrane tension using fluorescence lifetime imaging microscopy.

Fig. 4 and the legend for Fig. 5 have been updated.

Summary Secreted growth factors can act as morphogens that form spatial concentration gradients in developing organs, thereby controlling growth and patterning. For some morphogens, adaptation of the gradients to tissue size allows morphological patterns to remain proportioned as the organs grow. In the zebrafish pectoral fin, we found that BMP signaling forms a two-dimensional gradient. The length of the gradient scales with tissue length and its amplitude increases with fin size according to a power-law. Gradient scaling and amplitude power-laws are signatures of growth control by time derivatives of morphogenetic signaling: cell division correlates with the fold change over time of the cellular signaling levels. We show that Smoc1 regulates BMP gradient scaling and growth in the fin. Smoc1 scales the gradient by means of a feedback loop: Smoc1 is a BMP agonist and BMP signaling represses Smoc1 expression. Our work uncovers a layer of morphogen regulation during vertebrate appendage development., Graphical Abstract, Highlights • Zebrafish pectoral fins form two BMP signaling gradients that scale with fin size • The gradients show signatures of growth control by time derivatives of signaling • Smoc1 mutants fail to scale the BMP signaling gradient and have fin growth defects • A regulatory feedback loop Smoc1-BMP mediates BMP gradient scaling, The control of organ growth is essential for embryonic development. Mateus et al. show that during zebrafish pectoral fin development, two BMP morphogen signaling gradients form and expand proportionally to fin size, regulating growth. A feedback loop between Smoc1 (a conserved secreted factor) and BMP signaling mediates BMP gradient scaling.

We present a theory of pattern formation in growing domains inspired by biological examples of tissue development. Gradients of signaling molecules regulate growth, while growth changes these graded chemical patterns by dilution and advection. We identify a critical point of this feedback dynamics, which is characterized by spatially homogeneous growth and proportional scaling of patterns with tissue length. We apply this theory to the biological model system of the developing wing of the fruit fly \textit{Drosophila melanogaster} and quantitatively identify signatures of the critical point., 5 pages, 3 figures

How a developing organ grows and patterns to its final shape is an important question in developmental biology. Studies of growth and patterning in the Drosophila wing imaginal disc have identified a key player, the morphogen Decapentaplegic (Dpp). These studies provided insights into our understanding of growth control and scaling: expansion of the Dpp gradient correlated with the growth of the tissue. A recent report on growth of a Drosophila organ other than the wing, the eye imaginal disc, prompts a reconsideration of our models of growth control. Despite striking differences between the two, the Dpp gradient scales with the target tissues of both organs and the growth of both the wing and the eye is controlled by Dpp. The goal of this review is to discuss whether a parsimonious model of scaling and growth control can explain the relationship between the Dpp gradient and growth in these two different developmental systems.

Cell biologists now have tools and knowledge to generate useful quantitative data. But how can we make sense of these data, and are we measuring the correct parameters? Moreover, how can we test hypotheses quantitatively? To answer these questions, the theory of physics is required and is essential to the future of quantitative cell biology.

During asymmetric division, fate assignation in daughter cells is mediated by the partition of determinants from the mother. In the fly sensory organ precursor cell, Notch signalling partitions into the pIIa daughter. Notch and its ligand Delta are endocytosed into Sara endosomes in the mother cell and they are first targeted to the central spindle, where they get distributed asymmetrically to finally be dispatched to pIIa. While the processes of endosomal targeting and asymmetry are starting to be understood, the machineries implicated in the final dispatch to pIIa are unknown. We show that Sara binds the PP1c phosphatase and its regulator Sds22. Sara phosphorylation on three specific sites functions as a switch for the dispatch: if not phosphorylated, endosomes are targeted to the spindle and upon phosphorylation of Sara, endosomes detach from the spindle during pIIa targeting., Asymmetric segregation of cell fate determinants during cell division governs daughter cell fate. Here the authors show that Sara endosomes, known to regulate Notch signalling, are targeted to the mitotic spindle and once phosphorylated are asymmetrically dispatched into a daughter cell to determine cell fate.

The Biochemistry Department at the University of Geneva currently has four full professors, a professor emeritus, one assistant professor, two MER (Maître d'enseignement et de Recherche) and a permanent scientific collaborator. The research interests of the members of the Biochemistry Department are described.

During morphogenesis, organs grow to stereotyped sizes, but growth control mechanisms are poorly understood. Here, we measured the signaling dynamics of the morphogen Dpp, one of several Drosophila factors controlling morphogenetic growth, in the developing eye. In this tissue, the Dpp expression domain advances from the posterior to the anterior tissue edge. In front of this moving morphogen source, signaling inputs including Dpp activate the target gene hairy in a gradient that scales with tissue size. Proliferation, in turn, occurs in a mitotic wave in front of the source, whereas behind it, cells arrest and differentiate. We found that cells divide when their signaling levels have increased by around 60%. This simple mechanism quantitatively explains the proliferation and differentiation waves in wild type and mutants. Furthermore, this mechanism may be a common feature of different growth factors, because a Dpp-independent growth input also follows this growth rule.

The Notch signaling pathway plays important roles in many organisms and developmental contexts. The activities of the Notch receptor and of its ligand Delta are known to be regulated at several steps along the endocytic pathway. However, the precise molecular mechanism of Notch activation and the role played by endosomal sorting and trafficking remain elusive. We developed an antibody uptake assay to enable live imaging of endogenous internalized Notch and Delta in Drosophila tissues. In this chapter, we describe how to perform live antibody uptake assays in the Drosophila notum. In this tissue, Notch signaling plays a crucial role in the regulation of cell fate decisions in the lineage of sensory organ precursor (SOP) cells. We describe here how to do a correlative analysis of Notch/Delta live imaging in dividing SOPs and of the lineage of these particular SOPs. Combined with the wide range of genetic and chemical tools available in Drosophila research, these two methods will provide a better understanding of the role played by endocytic proteins and endosomal trafficking in Notch regulation, in terms of botch Notch trafficking and Notch signaling output.

Cells at different positions in a developing tissue receive different concentrations of signaling molecules, called morphogens, and this influences their cell fate. Morphogen concentration gradients have been proposed to control patterning as well as growth in many developing tissues. Some outstanding questions about tissue patterning by morphogen gradients are the following: What are the mechanisms that regulate gradient formation and shape? Is the positional information encoded in the gradient sufficiently precise to determine the positions of target gene domain boundaries? What are the temporal dynamics of gradients and how do they relate to patterning and growth? These questions are inherently quantitative in nature and addressing them requires measuring morphogen concentrations in cells, levels of downstream signaling activity, and kinetics of morphogen transport. Here we first present methods for quantifying morphogen gradient shape in which the measurements can be calibrated to reflect actual morphogen concentrations. We then discuss using fluorescence recovery after photobleaching to study the kinetics of morphogen transport at the tissue level. Finally, we present particle tracking as a method to study morphogen intracellular trafficking.

Oriented mitosis is essential during tissue morphogenesis. The Wnt/planar cell polarity (Wnt/PCP) pathway orients mitosis in a number of developmental systems, including dorsal epiblast cell divisions along the animal-vegetal (A-V) axis during zebrafish gastrulation. How Wnt signalling orients the mitotic plane is, however, unknown. Here we show that, in dorsal epiblast cells, anthrax toxin receptor 2a (Antxr2a) accumulates in a polarized cortical cap, which is aligned with the embryonic A-V axis and forecasts the division plane. Filamentous actin (F-actin) also forms an A-V polarized cap, which depends on Wnt/PCP and its effectors RhoA and Rock2. Antxr2a is recruited to the cap by interacting with actin. Antxr2a also interacts with RhoA and together they activate the diaphanous-related formin zDia2. Mechanistically, Antxr2a functions as a Wnt-dependent polarized determinant, which, through the action of RhoA and zDia2, exerts torque on the spindle to align it with the A-V axis.

In the wing imaginal disc of Drosophila melanogaster, the morphogen Dpp controls growth, probably in an instructive manner. Many models for growth control by Dpp have been proposed and have been extensively discussed elsewhere. In this review, we speculate on how instructive growth control could provide a link between Dpp signaling and cell growth and/or cell cycle progression and so implement morphogenetic growth control on the cellular and molecular levels.

A multitude of biological processes that involve multiple interaction partners are observed by two-color microscopy. Here we describe an analysis method for the robust quantification of correlation between signals in different color channels: particle image cross-correlation spectroscopy (PICCS). The method, which exploits the superior positional accuracy obtained in single-object and single-molecule microscopy, can extract the correlation fraction and length scale. We applied PICCS to correlation measurements in living tissues. The morphogen Decapentaplegic (Dpp) was imaged in wing imaginal disks of fruit fly larvae and we quantified what fraction of early endosomes contained Dpp.

Tissue morphogenesis depends on the spatial arrangement of cells during development. A number of mechanisms have been described to contribute to the final shape of a tissue or organ, ranging from cell intercalation to the response of cells to chemotactic cues. One such mechanism is oriented cell division. Oriented cell division is determined by the position of the mitotic spindle. Indeed, there is increasing evidence implicating spindle misorientation in tissue and organ misshaping, which underlies disease conditions such as tumorigenesis or polycystic kidneys. Here we review recent studies addressing how the direction of tissue growth is determined by the orientation of cell division and how both extrinsic and intrinsic cues control the position of the mitotic spindle.

Sizing Signals Growth regulation of the Drosophila wing imaginal disc critically depends on the Decapentaplegic (Dpp) morphogen gradient. How a graded Dpp signal is interpreted by cells to control homogeneous tissue growth remains unclear. Wartlick et al. (p. 1154 ; see the Perspective by Le Goff and Lecuit ) address this question by measuring the spatial and temporal changes of Dpp concentration and signaling activity during the disc growth phase and by quantifying cell proliferation parameters in the discs. Both modeling and experimental findings suggest that both Dpp concentration and signaling gradients scale with tissue size so that, on average, the onset of mitosis occurs when Dpp signaling levels have increased by 50% since the beginning of the cell cycle.

Border cell migration is a stereotyped migration occurring during the development of the Drosophila egg chamber. During this process, a cluster composed of six to eight follicle cells migrates between nurse cells toward the oocyte. Receptor tyrosine kinases (RTKs) are enriched at the leading edge of the follicle cells and establish the directionality of their migration. Endocytosis has been shown to play a role in the maintenance of this polarization; however, the mechanisms involved are largely unknown. In this study, we show that border cell migration requires the function of the small GTPases Rab5 and Rab11 that regulate trafficking through the early and the recycling endosome, respectively. Expression of a dominant negative form of rab11 induces a loss of the polarization of RTK activity, which correlates with a severe migration phenotype. In addition, we demonstrate that the exocyst component Sec15 is distributed in structures that are polarized during the migration process in a Rab11-dependent manner and that the down-regulation of different subunits of the exocyst also affects migration. Together, our data demonstrate a fundamental role for a plasma membrane–endosome trafficking cycle in the maintenance of active RTK at the leading edge of border cells during their migration.

Cell division involves a vast remodelling of cellular membranes. This is most apparent for the cell surface, but it is also true for internal vesicular organelles such as the Golgi apparatus. While the contribution of endocytosis to membrane trafficking and signal processing in interphase cells is well established, the role of the endocytic system in cell division has long been neglected. A number of recent studies have however shed novel light on this issue. Here, we review findings supporting the existence of two important links between endocytosis and mitosis: First, endocytic trafficking is essential to reshape the plasma membrane during cell division. Second, cell division affects the partitioning, the trafficking and hence the activity of the signalling molecules that are contained within endocytic compartments.

Morphogen concentration gradients provide positional information by activating target genes in a concentration-dependent manner. Recent reports show that the gradient of the syncytial morphogen Bicoid seems to provide precise positional information to determine target gene domains. For secreted morphogenetic ligands, the precision of the gradients, the signal transduction and the reliability of target gene expression domains have not been studied. Here we investigate these issues for the TGF-β-type morphogen Dpp. We first studied theoretically how cell-to-cell variability in the source, the target tissue, or both, contribute to the variations of the gradient. Fluctuations in the source and target generate a local maximum of precision at a finite distance to the source. We then determined experimentally in the wing epithelium: (1) the precision of the Dpp concentration gradient; (2) the precision of the Dpp signaling activity profile; and (3) the precision of activation of the Dpp target gene spalt. As captured by our theoretical description, the Dpp gradient provides positional information with a maximal precision a few cells away from the source. This maximal precision corresponds to a positional uncertainly of about a single cell diameter. The precision of the Dpp gradient accounts for the precision of the spaltexpression range, implying that Dpp can act as a morphogen to coarsely determine the expression pattern of target genes.

During development, cells acquire positional information by reading the concentration of morphogens. In the developing fly wing, a gradient of the transforming growth factor–β (TGF-β)–type morphogen decapentaplegic (Dpp) is transduced into a gradient of concentration of the phosphorylated form of the R-Smad transcription factor Mad. The endosomal protein Sara (Smad anchor for receptor activation) recruits R-Smads for phosphorylation by the type I TGF-β receptor. We found that Sara, Dpp, and its type I receptor Thickveins were targeted to a subpopulation of apical endosomes in the developing wing epithelial cells. During mitosis, the Sara endosomes and the receptors therein associated with the spindle machinery to segregate into the two daughter cells. Daughter cells thereby inherited equal amounts of signaling molecules and thus retained the Dpp signaling levels of the mother cell.

The analysis of membrane trafficking has in the past mainly dealt with single cells in culture. Recent studies of membrane trafficking in Drosophila focus on how cells are organized in tissues and form epithelia during embryogenesis. During these processes, the specific involvement of distinct biosynthetic and endocytic routes is starting to be understood. Once organized in epithelia, cells communicate with each other to make cell fate decisions through morphogen gradients and lateral inhibition. Endocytosis seems to play unexpected roles in shaping morphogen gradients and in biasing lateral inhibition events. Once committed to a developmental program, cells differentiate. In the case of neurons, trafficking through the biosynthetic and endocytic pathways may give the necessary speed of response and versatility to axons that navigate through a changing environment during pathfinding.

The ability to internalize macromolecules by endocytosis is a property of all eukaryotic cells. Frontline research on endocytosis has been presented in a successful series of biannual meetings in Europe. This year's meeting on "Membrane Dynamics in Endocytosis" was held September 13-18 in Acquafredda di Maratea, on the coast of southern Italy. Four key questions were addressed: What are the molecular mechanisms of endocytic membrane trafficking? How does endocytosis modulate receptor signaling and vice versa? What is the importance of endocytosis during development? How do endocytic organelles contribute to immunity or susceptibility to pathogens?

Nowadays, the focus of developmental studies is shifting away from formal models of developmental pathways that are characterised by flow charts of controlling factors connected by arrows, to mechanistic models that explain developmental processes at the cellular level. Surprisingly, this shift towards a cellular view of developmental biology is occurring simultaneously across a range of model organisms. One consequence of taking such a cell biological view of development is that many model organisms are now becoming good models for studies of human disease and therapy.

During the last decade, many of the factors and mechanisms controlling membrane and protein trafficking in general and endocytic trafficking in particular have been uncovered. We have a detailed understanding of the different endocytic trafficking steps: plasma membrane budding, endocytic vesicle motility and fusion with the endosome, recycling, transcytosis and lysosomal degradation. The kinetics and trafficking pathway of many signaling receptors and the relevance of endocytic trafficking during signaling in many mammalian cultured cells are also well understood. However, only in recent years has the role of endocytic trafficking during cell-to-cell communication during development, i.e. during patterning, induction and lateral inhibition, begun to be explored. The contribution of Drosophila developmental genetics and cell biology has been fundamental in elucidating the essential role of endocytosis during these processes. Reviewed here are some of the recent developments on the role of endocytic trafficking during long- and short-range signaling and during lateral inhibition.

Morphogens are secreted signaling molecules which form spatial concentration gradients while moving away from a restricted source of production. A simple model of gradient formation postulates that the morphogens dilute as they diffuse between cells. In this review we discuss recent data supporting the idea that movement of the morphogen could also occur via vesicular trafficking through the cells. We explore the implications of these results for the control of gradient formation and the determination of the gradient slope which ultimately encodes the coordinates of positional information.

During asymmetric division, fate determinants at the cell cortex segregate unequally into the two daughter cells. It has recently been shown that Sara (Smad anchor for receptor activation) signalling endosomes in the cytoplasm also segregate asymmetrically during asymmetric division1,2. Biased dispatch of Sara endosomes mediates asymmetric Notch/Delta signalling during the asymmetric division of sensory organ precursors in Drosophila1. In flies, this has been generalized to stem cells in the gut3 and the central nervous system1, and, in zebrafish, to neural precursors of the spinal cord4. However, the mechanism of asymmetric endosome segregation is not understood. Here we show that the plus-end kinesin motor Klp98A targets Sara endosomes to the central spindle, where they move bidirectionally on an antiparallel array of microtubules. The microtubule depolymerizing kinesin Klp10A and its antagonist Patronin generate central spindle asymmetry. This asymmetric spindle, in turn, polarizes endosome motility, ultimately causing asymmetric endosome dispatch into one daughter cell. We demonstrate this mechanism by inverting the polarity of the central spindle by polar targeting of Patronin using nanobodies (single-domain antibodies). This spindle inversion targets the endosomes to the wrong cell. Our data uncover the molecular and physical mechanism by which organelles localized away from the cellular cortex can be dispatched asymmetrically during asymmetric division.

The Notch signaling pathway plays important roles in many organisms and developmental contexts. The activities of the Notch receptor and of its ligand Delta are known to be regulated at several steps along the endocytic pathway. However, the precise molecular mechanism of Notch activation and the role played by endosomal sorting and trafficking remain elusive. We developed an antibody uptake assay to enable live imaging of endogenous internalized Notch and Delta in Drosophila tissues. In this chapter, we describe how to perform live antibody uptake assays in the Drosophila notum. In this tissue, Notch signaling plays a crucial role in the regulation of cell fate decisions in the lineage of sensory organ precursor (SOP) cells. We describe here how to do a correlative analysis of Notch/Delta live imaging in dividing SOPs and of the lineage of these particular SOPs. Combined with the wide range of genetic and chemical tools available in Drosophila research, these two methods will provide a better understanding of the role played by endocytic proteins and endosomal trafficking in Notch regulation, in terms of botch Notch trafficking and Notch signaling output.

Secreted morphogens such as the Drosophila TGF-β homolog Decapentaplegic (Dpp) are thought to spread through target tissues and form long-range concentration gradients providing positional information. Using a GFP-Dpp fusion, we monitored a TGF-β family member trafficking in situ throughout the target tissue and forming a long-range concentration gradient. Evidence is presented that long-range Dpp movement involves Dpp receptor and Dynamin functions. We also show that the rates of endocytic trafficking and degradation determine Dpp signaling range. We propose a model where the gradient is formed via intracellular trafficking initiated by receptor-mediated endocytosis of the ligand in receiving cells with the gradient slope controlled by endocytic sorting of Dpp toward recycling versus degradation.

The stomatogastric nervous system (SNS) of Drosophila is a simply organized neural circuitry that innervates the anterior enteric system. Unlike the central and the peripheral nervous systems, the SNS derives from a compact epithelial anlage in which three invagination centers, each giving rise to an invagination fold headed by a tip cell, are generated. Tip cell selection involves lateral inhibition, a process in which Wingless (Wg) activity adjusts the range of Notch signaling. Here we show that RTK signaling mediated by the Drosophila homolog of the epidermal growth factor receptor, DER, plays a key role in two consecutive steps during early SNS development. Like Wg, DER signaling participates in adjusting the range of Notch-dependent lateral inhibition during tip cell selection. Subsequently, tip cells secrete the DER ligand Spitz and trigger local RTK signaling, which initiates morphogenetic movements resulting in the tip cell-directed invaginations within the SNS anlage.

Pattern formation along the anterior-posterior (A/P) axis of the developing Drosophila wing depends on Decapentaplegic (Dpp), a member of the conserved transforming growth factor b (TGFb ) family of secreted proteins. Dpp is expressed in a stripe along the A/P compartment boundary of the wing imaginal disc and forms a long-range concentration gradient with morphogen-like properties which generates distinct cell fates along the A/P axis. We have monitored Dpp expression and Dpp signalling in endocytosis-mutant wing imaginal discs which develop severe pattern defects specifically along the A/P wing axis. The results show that the size of the Dpp expression domain is expanded in endocytosis-mutant wing discs. However, this expansion did not result in a concommittant expansion of the functional range of Dpp activity but rather its reduction as indicated by the reduced expression domain of the Dpp target gene spalt. The data suggest that clathrinmediated endocytosis, a cellular process necessary for membrane recycling and vesicular trafficking, participates in Dpp action during wing development. Genetic interaction studies suggest a link between the Dpp receptors and clathrin. Impaired endocytosis does not interfere with the reception of the Dpp signal or the intracellular processing of the mediation of the signal in the responder cells, but rather affects the secretion and/or the distribution of Dpp in the developing wing cells. q 1999 Elsevier Science Ireland Ltd. All rights reserved.

Three recent papers in Developmental Cell (by Yamamoto et al., Choi et al., and Sigismund et al.) show that the endocytic route of internalization can determine whether signaling receptors are degraded or recycled, and whether they initiate signaling from an endosomal platform. However, the same route can serve opposite functions in different signaling pathways: there is no "unifying theory" of trafficking.

NOTCH signaling is a developmental pathway known to play critical roles during embryonic development as well as for the regulation of self-renewing tissues. Aberrant activation of NOTCH signaling leads to deregulation of the self-renewal process resulting in sustained proliferation, evasion of cell death, loss of differentiation capacity, invasion and metastasis, all of which are hallmarks of cancer. Over activation of NOTCH in human cancers can be a consequence of over expression of NOTCH ligands/receptors, GOF mutations in NOTCH receptors as well as chromosomal translocations leading to constitutive activation of the pathway. Given the importance of Notch signaling in human cancers, several therapeutic approaches have been utilized to block NOTCH signaling. Two of these strategies are; a) the use of monoclonal blocking antibodies (Mabs) against NOTCH ligands and receptors and b) the use of small molecule γ-secretase inhibitors (GSIs). However, these approaches can only be effective if tumor cells express full-length ligand or receptor molecules. On the contrary, in human cancers harbouring NOTCH gene fusion due to chromosomal translocations, the use of Mabs and GSIs will have very limited clinical benefits. A third, yet not fully explored approach could be the blockage of NOTCH signalling by targeting the most downstream event in the NOTCH cascade i,e NOTCH transcriptional activation complex using small molecule inhibitors. Here we report discovery and identification of a novel, orally-active small molecule inhibitor, named CB-103, of the NOTCH pathway that blocks NOTCH signaling by targeting the NOTCH transcriptional activation complex in the nucleus. CB-103 has shown the ability to block NOTCH signalling in human cancer cell lines, induce neurogenic phenotype in drosophila, induce muscle cell differentiation and inhibit NOTCH dependent cellular processes in mice. In addition, CB-103 exhibit anti-tumor efficacy in a xenograft model of human triple negative breast cancer resistant to GSIs and Mabs against NOTCH ligands/receptors. Furthermore, CB-103 has shown a remarkable activity in PDX models of human T-ALL harbouring activation of the NOTCH pathway. Additional studies are underway on several analogs of CB-103 to determine ADME/PK/PD profile and nominate a development candidate for further clinical development of this novel inhibitor of the NOTCH pathway. Citation Format: Rajwinder Lehal, Viktoras Frismantas, Sylvain Loubéry, Viktoria Reinmuller, Gerardo Turcatti, Marcos Gonzalez-Gaitan, Jean-Pierre Bourquin, Freddy Radtke. A novel small molecule inhibitor of the Notch transcription activation complex. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 338.

Because signaling mediated by the transcription factor nuclear factor κB (NF-κB) is initiated by ligands and receptors that can undergo internalization, we investigated how endocytic trafficking regulated this key physiological pathway. We depleted all of the ESCRT (endosomal sorting complexes required for transport) subunits, which mediate receptor trafficking and degradation, and found that the components Tsg101, Vps28, UBAP1, and CHMP4B were essential to restrict constitutive NF-κB signaling in human embryonic kidney 293 cells. In the absence of exogenous cytokines, depletion of these proteins led to the activation of both canonical and noncanonical NF-κB signaling, as well as the induction of NF-κB-dependent transcriptional responses in cultured human cells, zebrafish embryos, and fat bodies in flies. These effects depended on cytokine receptors, such as the lymphotoxin β receptor (LTβR) and tumor necrosis factor receptor 1 (TNFR1). Upon depletion of ESCRT subunits, both receptors became concentrated on and signaled from endosomes. Endosomal accumulation of LTβR induced its ligand-independent oligomerization and signaling through the adaptors TNFR-associated factor 2 (TRAF2) and TRAF3. These data suggest that ESCRTs constitutively control the distribution of cytokine receptors in their ligand-free state to restrict their signaling, which may represent a general mechanism to prevent spurious activation of NF-κB.

Morphogen gradients regulate the patterning and growth of many tissues, hence a key question is how they are established and maintained during development. Theoretical descriptions have helped to explain how gradient shape is controlled by the rates of morphogen production, spreading and degradation. These effective rates have been measured using fluorescence recovery after photobleaching (FRAP) and photoactivation. To unravel which molecular events determine the effective rates, such tissue-level assays have been combined with genetic analysis, high-resolution assays, and models that take into account interactions with receptors, extracellular components and trafficking. Nevertheless, because of the natural and experimental data variability, and the underlying assumptions of transport models, it remains challenging to conclusively distinguish between cellular mechanisms.

Optical highlighters are photoactivatable fluorescent molecules that exhibit pronounced changes in their spectral properties in response to irradiation with light of a specific wavelength and intensity. Here, we present a novel design strategy for a new class of caged BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) fluorophores, based on the use of photoremovable protecting groups (PRPGs) with high reduction potentials that serve as both a photosensitive unit and a fluorescence quencher via photoinduced electron transfer (PeT). 2,6-Dinitrobenzyl (DNB)-caged BODIPY was efficiently photoactivated, with activation ratios exceeding 600-fold in aqueous solutions. We then combined this photoactivatable fluorophore with a SNAP (mutant of O(6)-alkylguanine DNA alkyltransferase) ligand to obtain a small-molecule-based optical highlighter for visualization of protein dynamics, using the well-established SNAP tag technology. As proof of concept, we demonstrate spatiotemporal imaging of the fusion protein of epidermal growth factor receptor (EGFR) with SNAP tag in living cells. We also demonstrate highlighting of cells of interest in live zebrafish embryos, using the fusion protein of histone 2A with SNAP tag.

Developmental gene functions of Drosophila are typically characterized by a recognizable mutant phenotype. When molecular probes of such genes were used to isolate homologues, distinct spatially and temporally restricted expression patterns were observed in vertebrates as well. However, corresponding "gene knock-outs" often revealed subtle or no scorable phenotypes, a phenomenon attributed to redundant gene functions. We found that the evolutionarily related genes knirps (kni) and knirps-related (knrl) contribute to a similar phenomenon in Drosophila. The two closely situated genes show identical expression patterns in the developing embryo, including the posterior and anterior expression domains in the blastoderm. Here we show that the two biochemically equivalent gene products are both functional in the head anlage and that the lack of one gene activity can be overcome by the activity of the other. Whereas kni is also required for abdominal segmentation, knrl is nonfunctional in its posterior expression domain. Thus, the kni/knrl pair of genes provides a region-specific buffering system, rather than a case of global functional redundancy.

The development of the insect head tagma involves massive rearrangements and secondary fusions of segment anlagen during embryogenesis. Due to the lack of reliable morphological markers, the number, identity, and sequence of the head segments, particularly in the pregnathal region, are still a matter of ongoing debates. We examined the complex array of internal structures of the embryonic Drosophila melanogaster head such as the sensory structures and nerves of the peripheral and stomatogastric nervous systems, and we used embryonic head mutations causing a lack of overlapping segment anlagen to unravel the segmental identity and the sequence of the neural elements. Our results provide evidence for seven distinct segments in the Drosophila head, each characterized by a specific set of sensory neurons, consistent with the proposal that insects, myriapods, and crustaceans share a monophyletic evolutionary tree from a common annelid-like ancestor.

Morphogenetic processes, based on the temporal and spatial control of cell proliferation, are involved in determining the size and shape of an organism. We have used clonal analysis, employing X-ray-induced mitotic recombination, to study cell proliferation and differentiation processes in the developing wing imaginal disc of Drosophila. Our results show a non-uniform distribution of mitotic activities during different stages of wing development. This may reflect waves of cell proliferation which derive from distinct centers of cell proliferation within the growing wing imaginal disc. These proliferation centers are located within the major wing compartments (i.e. the anterior, posterior, dorsal and ventral compartments) and they are restricted to the areas which give rise to the intervein regions of the adult wing. The mitotic recombination analysis, combined with the study of Minute and gynandromorph mosaics, show that the presumptive vein regions of the wing represent distinct boundaries which delimit the proliferation centers to the intervein regions. We present a generative model of wing morphogenesis that is consistent with our results.

The region specific homeotic gene spalt (sal) of Drosophila melanogaster promotes the specification of terminal pattern elements as opposed to segments in the trunk. Our results show that the previously reported sal transcription unit was misidentified. Based on P-element mediated germ line transformation and DNA sequence analysis of sal mutant alleles, we identified the transcription unit that carries sal function. sal is located close to the misidentified transcription unit, and it is expressed in similar temporal and spatial patterns during embryogenesis. The sal gene encodes a zinc finger protein of novel structure composed of three widely spaced 'double zinc finger' motifs of internally conserved sequences and a single zinc finger motif of different sequence. Antibodies produced against the sal protein show that sal is first expressed at the blastoderm stage and later in restricted areas of the embryonic nervous system as well as in the developing trachea. The antibodies detect sal homologous proteins in corresponding spatial and temporal patterns in the embryos of related insect species. Sequence analysis of the sal gene of Drosophila virilis, a species which is phylogenetically separated by approximately 60 million years, suggests that the sal function is conserved during evolution, consistent with its proposed role in head formation during arthropod evolution.

Morphogens are secreted signalling molecules that control the patterning and growth of developing organs. How morphogens regulate patterning is fairly well understood; however, how they control growth is less clear. Four principal models have been proposed to explain how the morphogenetic protein Decapentaplegic (DPP) controls the growth of the wing imaginal disc in the fly. Recent studies in this model system have provided a wealth of experimental data on growth and DPP gradient properties, as well as on the interactions of DPP with other signalling pathways. These findings have allowed a more precise formulation and evaluation of morphogenetic growth models. The insights into growth control by the DPP gradient will also be useful for understanding other morphogenetic growth systems.

This Podcast features a conversation with the authors of a Research Article published in the 4 March 2011 issue of Science . Marcos Gonzalez-Gaitan and Frank Julicher discuss their mathematical description of how the graded concentration of the morphogen Dpp induces homogeneous cellular proliferation across the fly wing and haltere imaginal discs. They propose a mechanism by which changes in activation of the Dpp signaling pathway over time account for the observed growth of discs.

We present a method for the specific labelling of fusion proteins with synthetic fluorophores in Zebrafish. The method uses the SNAP-tag technology and O-6-benzylguanine derivatives of various synthetic fluorophores. We demonstrate how the method can be used to label subcellular structures in Zebrafish such as the nucleus, cell membranes, and endosomal membranes. The stability of the synthetic fluorophores makes them attractive choices for long-term imaging and allows, unlike most of the autofluorescent proteins, the use of acid fixatives such as trichloroacetic acid. Furthermore, the use of O-6-benzylguanine derivatives bearing caged fluorescein allows cell lineage tracing through photo-deprotection of the fluorophore and its detection either through fluorescence microscopy or through immunohistochemistry after fixation using anti-fluorescein antibodies. Developmental Dynamics 240:820-827, 2011. (C) 2011 Wiley-Liss, Inc.

In addition to apicobasal polarization, some epithelia also display polarity within the plane of the epithelium. To what extent polarized endocytosis plays a role in the establishment and maintenance of planar cell polarity (PCP) is at present unclear. Here, we investigated the role of Rabenosyn-5 (Rbsn-5), an evolutionarily conserved effector of the small GTPase Rab5, in the development of Drosophila wing epithelium. We found that Rbsn-5 regulates endocytosis at the apical side of the wing epithelium and, surprisingly, further uncovered a novel function of this protein in PCP. At early stages of pupal wing development, the PCP protein Fmi redistributes between the cortex and Rab5- and Rbsn-5-positive early endosomes. During planar polarization, Rbsn-5 is recruited at the apical cell boundaries and redistributes along the proximodistal axis in an Fmi-dependent manner. At pre-hair formation, Rbsn-5 accumulates at the bottom of emerging hairs. Loss of Rbsn-5 causes intracellular accumulation of Fmi and typical PCP alterations such as defects in cell packing, in the polarized distribution of PCP proteins, and in hair orientation and formation. Our results suggest that establishment of planar polarity requires the activity of Rbsn-5 in regulating both the endocytic trafficking of Fmi at the apical cell boundaries and hair morphology.

The tissues of a developing embryo are simultaneously patterned, moved and differentiated according to an exchange of information between their constituent cells. We argue that these complex self-organizing phenomena can only be fully understood with quantitative mathematical frameworks that allow specific hypotheses to be formulated and tested. The quantitative and dynamic imaging of growing embryos at the molecular, cellular and tissue level is the key experimental advance required to achieve this interaction between theory and experiment. Here we describe how mathematical modelling has become an invaluable method to integrate quantitative biological information across temporal and spatial scales, serving to connect the activity of regulatory molecules with the morphological development of organisms.

Many developmental processes of multicellular organisms involve the patterning and growth of two-dimensional tissues, so called epithelia. We have quantified the growth of the wing imaginal disk, which is the precursor of the adult wing, of the fruit fly Drosophila melanogaster. We find that growth follows a simple rule with exponentially decreasing area growth rate. Anisotropies of growth can be precisely determined by comparing experimental results to a continuum theory. Growth anisotropies are to good approximation constant in space and time. They are weak in wild-type wing disks but threefold increased in GFP-Dpp disks in which the morphogen Dpp is overexpressed. Our findings indicate that morphogens such as Dpp control tissue shape via oriented cell divisions that generate anisotropic growth.

Endocytosis has a crucial role during Notch signalling after the asymmetric division of fly sensory organ precursors (SOPs): directional signalling is mediated by differential endocytosis of the ligand Delta and the Notch effector Sanpodo in one of the SOP daughters, pIIb. Here we show a new mechanism of directional signalling on the basis of the trafficking of Delta and Notch molecules already internalized in the SOP and subsequently targeted to the other daughter cell, pIIa. Internalized Delta and Notch traffic to an endosome marked by the protein Sara. During SOP mitosis, Sara endosomes containing Notch and Delta move to the central spindle and then to pIIa. Subsequently, in pIIa (but not in pIIb) Notch appears cleaved in Sara endosomes in a gamma-secretase- and Delta internalization-dependent manner, indicating that the release of the intracellular Notch tail to activate Notch target genes has occurred. We thus uncover a new mechanism to bias signalling even before asymmetric endocytosis of Sanpodo and Delta takes place in the daughter cells: already during SOP mitosis, asymmetric targeting of Delta and Notch-containing Sara endosomes will increase Notch signalling in pIIa and decrease it in pIIb.

The clathrin heavy chain is a fundamental element in endocytosis and therefore, in the internalization of several cell-surface receptors through which cells interact with their environment. Here we show that the only non-lethal mutant allele of the clathrin heavy chain identified to date in metazoans, the Drosophila Chc4, involves the substitution of a residue at the knee region of the molecule that impairs clathrin-dependent endocytosis. We have investigated the consequences of this endocytic defect in Drosophila retinal development and found that it produces an inhibition of programmed cell death in the retinal lattice, followed by widespread death of interommatidial pigment cells once retinal development has been completed. Through genetic interactions and transgenic analyses, we show that Chc4 phenotypes are caused by a Notch receptor gain-of-function, providing a dramatic example of the importance of Notch down-regulation by endocytosis. An increase in Notch signaling is also observed in Drosophila wings in response to the mutant clathrin, suggesting that Notch levels are controlled by clathrin-dependent endocytosis. We discuss the implications of these findings for current models on eye-development and for the role of endocytosis in Notch signaling., This work was supported by the Spanish Ministry of Education and Sciences research grants BFI2001-3887 and SAF2004-06408 to J.V. and F.M., and doctorate Grant FPI2001-1561 to S.P.