Nerve growth factor (NGF) stimulation of cholinergic telencephalic neurons in aggregating cell cultures.

The addition of nerve growth factor (2.5S NGF) to serum-free aggregating cell cultures of fetal rat telencephalon greatly stimulated the developmental increase in choline acetyltransferase activity. Two other neuronal enzymes, acetylcholinesterase and glutamic acid decarboxylase, showed only slightly increased activities after NGF treatment whereas the total protein content of the cultures and the activity of 2',3'- cyclic nucleotide phosphodiesterase remained unchanged. The stimulation of choline acetyltransferase was dependent on the NGF media concentrations, showing a 50% maximum effect (120% increase) at approximately 3 ng/ml (10-10 M 2.5S NGF). NGF treatments during different culture periods showed that the cholinergic neurons remained responsive for at least 19 days. The continued treatment was the most effective; however, an initial treatment for only 5 days still caused a significant stimulation of choline acetyltransferase on day 19. The observed stimulation appeared to be specific to NGF. Univalent antibody fragments (Fab) against 2.5S NGF completely abolished the NGF-dependent increase in choline acetyltransferase activity, whereas Fab fragments of control IgG were ineffective. Furthermore, angiotensin II, added in high amounts to the cultures, showed no stimulatory effect. The present results suggest that certain populations of rat brain neurons are responsive to nerve growth factor.

Phototropism: Translating light into directional growth.

Phototropism allows plants to align their photosynthetic tissues with incoming light. The direction of incident light is sensed by the phototropin family of blue light photoreceptors (phot1 and phot2 in Arabidopsis), which are light-activated protein kinases. The kinase activity of phototropins and phosphorylation of residues in the activation loop of their kinase domains are essential for the phototropic response. These initial steps trigger the formation of the auxin gradient across the hypocotyl that leads to asymmetric growth. The molecular events between photoreceptor activation and the growth response are only starting to be elucidated. In this review, we discuss the major steps leading from light perception to directional growth concentrating on Arabidopsis. In addition, we highlight links that connect these different steps enabling the phototropic response.

PGE2-induced colon cancer growth is mediated by mTORC1

La mortalité du cancer colorectal reste importante malgré les nombreux progrès effectués dans la compréhension des mécanismes responsables à son développement. Dans ce contexte, il a été démontré qu'une enzyme appelée cyclooxygénase-2 (COX-2) joue un rôle important dans la pathogenèse du cancer colorectal. En effet, les métabolites produits par cette enzyme, en particulier la Prostaglandine E2 (PGE2), sont capables de directement stimuler la prolifération et la survie des cellules tumorales nécessaires à la progression tumorale. De plus, la PGE2 stimule également la formation de nouveaux vaisseaux sanguins indispensable à la croissance tumorale en induisant la formation du facteur de croissance vasculaire (VEGF). L'importance de COX- 2 dans le cancer colorectal ne se limite pas au niveau expérimental mais a aussi été démontré chez des patients où il a été prouvé que des inhibiteurs chimiques de COX-2 comme l'aspirine réduisaient le risque de développer un cancer colorectal. Il est donc important de caractériser et de comprendre les mécanismes par lesquels la COX-2 et les PGE2 participent au développement du cancer colorectal afin de générer de nouvelles approches thérapeutiques. Dans cette étude, nous avons observé qu'un complexe protéique intracellulaire appelé mTORC1 joue un rôle important dans la prolifération de cellules du cancer colorectal induite par la PGE2. En effet, nous avons trouvé que l'activité de mTORC1 était augmentée après stimulation des cellules tumorales par la PGE2. Nous avons également trouvé que cette stimulation était médiée par un type spécifique de récepteurs de la PGE2 appelé EP4. L'inhibition de mTORC1 par des composés chimiques ou par interférence de RNA bloque la prolifération cellulaire induite par la PGE2. De même, la production du facteur de croissance endothéliale (VEGF) par la PGE2 est bloquée par les inhibiteurs de mTORC1. Nos résultats montrent donc que mTORC1 est un intermédiaire cellulaire important dans la croissance tumorale induite par la PGE2 ainsi que dans la production de VEGF. mTORC1 représente de ce fait une cible thérapeutique intéressante dans le cancer colorectal qui mérite d'être évaluée dans des études cliniques.

Igneous layering, fractional crystallization and growth of granitic plutons: The Dolbel batholith in SW Niger

This study reassesses the development of compositional layering during the growth of granitic plutons, with emphasis on fractional crystallization and its interaction with both injection and inflation-related deformation. The Dolbel batholith (SW Niger) consists of 14, kilometre-sized plutons emplaced by pulsed magma inputs. Each pluton has a coarse-grained core and a peripheral layered series. Rocks consist of albite (An(<= 11)), K-feldspar (Or(96 99), Ab(1) (4)), quartz, edenite (X(Mg)=0337-0.55), augite (X(Mg)=0.65-0.72) and accessories (apatite, titanite and Fe-Ti-oxides). Whole-rock compositions are metaluminous, sodic (K(2)O/Na(2)O=0.49-0.62) and iron-rich [FeO(tot)/(FeO(tot)+MgO)=0.65-0.82]. The layering is present as size-graded and modally graded, sub-vertical, rhythmic units. Each unit is composed of three layers, which are, towards the interior: edenite +/- plagioclase (C(a/p)), edenite+plagioclase+augite+quartz (C(q)), and edenite+plagioclase+augite+quartz+K-feldspar (C(k)). All phases except quartz show zoned microstructures consisting of external intercumulus overgrowths, a central section showing oscillatory zoning and, in the case of amphibole and titanite, complexly zoned cores. Ba and Sr contents of feldspars decrease towards the rims. Plagioclase crystal size distributions are similar in all units, suggesting that each unit experienced a similar thermal history. Edenite, characteristic of the basal C(a/p) layer, is the earliest phase to crystallize. Microtextures and phase diagrams suggest that edenite cores may have been brought up with magma batches at the site of emplacement and mechanically segregated along the crystallized wall, whereas outer zones of the same crystals formed in situ. The subsequent C(q) layers correspond to cotectic compositions in the Qz-Ab-Or phase diagram at P(H2O)=5 kbar. Each rhythmic unit may therefore correspond to a magma batch and their repetition to crystallization of recurrent magma recharges. Microtextures and chemical variations in major phases allow four main crystallization stages to be distinguished: (1) open-system crystallization in a stirred magma during magma emplacement, involving dissolution and overgrowth (core of edenite and titanite crystals); (2) in situ fractional crystallization in boundary layers (C(a/p) and C(q) layers); (3) equilibrium `en masse' eutectic crystallization (C(k) layers); (4) compaction and crystallization of the interstitial liquid in a highly crystallized mush (e. g. feldspar intercumulus overgrowths). It is concluded that the formation of the layered series in the Dolbel plutons corresponds principally to in situ differentiation of successive magma batches. The variable thickness of the Ck layers and the microtextures show that crystallization of a rhythmic unit stops and it is compacted when a new magma batch is injected into the chamber. Therefore, assembly of pulsed magma injections and fractional crystallization are independent, but complementary, processes during pluton construction.

An SH2 domain-containing 5' inositolphosphatase inhibits insulin-induced GLUT4 translocation and growth factor-induced actin filament rearrangement.

Tyrosine kinase receptors lead to rapid activation of phosphatidylinositol 3-kinase (PI3 kinase) and the subsequent formation of phosphatidylinositides (PtdIns) 3,4-P2 and PtdIns 3,4, 5-P3, which are thought to be involved in signaling for glucose transporter GLUT4 translocation, cytoskeletal rearrangement, and DNA synthesis. However, the specific role of each of these PtdIns in insulin and growth factor signaling is still mainly unknown. Therefore, we assessed, in the current study, the effect of SH2-containing inositol phosphatase (SHIP) expression on these biological effects. SHIP is a 5' phosphatase that decreases the intracellular levels of PtdIns 3,4,5-P3. Expression of SHIP after nuclear microinjection in 3T3-L1 adipocytes inhibited insulin-induced GLUT4 translocation by 100 +/- 21% (mean +/- the standard error) at submaximal (3 ng/ml) and 64 +/- 5% at maximal (10 ng/ml) insulin concentrations (P < 0.05 and P < 0.001, respectively). A catalytically inactive mutant of SHIP had no effect on insulin-induced GLUT4 translocation. Furthermore, SHIP also abolished GLUT4 translocation induced by a membrane-targeted catalytic subunit of PI3 kinase. In addition, insulin-, insulin-like growth factor I (IGF-I)-, and platelet-derived growth factor-induced cytoskeletal rearrangement, i.e., membrane ruffling, was significantly inhibited (78 +/- 10, 64 +/- 3, and 62 +/- 5%, respectively; P < 0.05 for all) in 3T3-L1 adipocytes. In a rat fibroblast cell line overexpressing the human insulin receptor (HIRc-B), SHIP inhibited membrane ruffling induced by insulin and IGF-I by 76 +/- 3% (P < 0.001) and 68 +/- 5% (P < 0.005), respectively. However, growth factor-induced stress fiber breakdown was not affected by SHIP expression. Finally, SHIP decreased significantly growth factor-induced mitogen-activated protein kinase activation and DNA synthesis. Expression of the catalytically inactive mutant had no effect on these cellular responses. In summary, our results show that expression of SHIP inhibits insulin-induced GLUT4 translocation, growth factor-induced membrane ruffling, and DNA synthesis, indicating that PtdIns 3,4,5-P3 is the key phospholipid product mediating these biological actions.

Reduction of choroidal neovascularization in mice by adeno-associated virus-delivered anti-vascular endothelial growth factor short hairpin RNA.

BACKGROUND: Strategies leading to the long-term suppression of inappropriate ocular angiogenesis are required to avoid the need for repetitive monthly injections for treatment of diseases of the eye, such as age-related macular degeneration (AMD). The present study aimed to develop a strategy for the sustained repression of vascular endothelial growth factor (VEGF), which is identified as the key player in exudative AMD. METHODS: We have employed short hairpin (sh)RNAs combined with adeno-associated virus (AAV) delivery to obtain the targeted expression of potent gene-regulatory molecules. Anti-VEGF shRNAs were analyzed in human retinal pigment epithelial (RPE) cells using Renilla luciferase screening. For in vivo delivery of the most potent shRNA, self-complementary AAV vectors were packaged in serotype 8 capsids (scAAV2/8-hU6-sh9). In vivo efficacy was evaluated either by injection of scAAV2/8-hU6-sh9 into murine hind limb muscles or in a laser-induced murine model of choroidal neovascularization (CNV) following scAAV2/8-hU6-sh9 subretinal delivery. RESULTS: Plasmids encoding anti-VEGF shRNAs showed efficient knockdown of human VEGF in RPEs. Intramuscular administration led to localized expression and 91% knockdown of endogenous murine (m)VEGF. Subsequently, the ability of AAV2/8-encoded shRNAs to impair vessel formation was evaluated in the murine model of CNV. In this model, the sizes of the CNV were significantly reduced (up to 48%) following scAAV2/8-hU6-sh9 subretinal delivery. CONCLUSIONS: Using anti-VEGF vectors, we have demonstrated efficient silencing of endogenous mVEGF and showed that subretinal administration of scAAV2/8-hU6-sh9 has the ability to impair vessel formation in an AMD animal model. Thus, AAV-encoded shRNA can be used for the inhibition of neovascularization, leading to the development of sustained anti-VEGF therapy. Copyright © 2012 John Wiley & Sons, Ltd.

Prostaglandin E(2) promotes colorectal adenoma growth via transactivation of the nuclear peroxisome proliferator-activated receptor delta.

Cyclooxygenase-derived prostaglandin E(2) (PGE(2)) is the predominant prostanoid found in most colorectal cancers (CRC) and is known to promote colon carcinoma growth and invasion. However, the key downstream signaling pathways necessary for PGE(2)-induced intestinal carcinogenesis are unclear. Here we report that PGE(2) indirectly transactivates PPARdelta through PI3K/Akt signaling, which promotes cell survival and intestinal adenoma formation. We also found that PGE(2) treatment of Apc(min) mice dramatically increased intestinal adenoma burden, which was negated in Apc(min) mice lacking PPARdelta. We demonstrate that PPARdelta is a focal point of crosstalk between the prostaglandin and Wnt signaling pathways which results in a shift from cell death to cell survival, leading to increased tumor growth.

GacS sensor domains pertinent to the regulation of exoproduct formation and to the biocontrol potential of Pseudomonas fluorescens CHA0.

In the root-colonizing biocontrol strain CHA0 of Pseudomonas fluorescens, cell density-dependent synthesis of extracellular, plant-beneficial secondary metabolites and enzymes is positively regulated by the GacS/GacA two-component system. Mutational analysis of the GacS sensor kinase using improved single-copy vectors showed that inactivation of each of the three conserved phosphate acceptor sites caused an exoproduct null phenotype (GacS-), whereas deletion of the periplasmic loop domain had no significant effect on the expression of exoproduct genes. Strain CHA0 is known to synthesize a solvent-extractable extracellular signal that advances and enhances the expression of exoproduct genes during the transition from exponential to stationary growth phase when maximal exoproduct formation occurs. Mutational inactivation of either GacS or its cognate response regulator GacA abolished the strain's response to added signal. Deletion of the linker domain of the GacS sensor kinase caused signal-independent, strongly elevated expression of exoproduct genes at low cell densities. In contrast to the wild-type strain CHA0, the gacS linker mutant and a gacS null mutant were unable to protect tomato plants from crown and root rot caused by Fusarium oxysporum f. sp. radicis-lycopersici in a soil-less microcosm, indicating that, at least in this plant-pathogen system, there is no advantage in using a signal-independent biocontrol strain.

The Schizosaccharomyces pombe cdc14 gene is required for septum formation and can also inhibit nuclear division.

A conditional heat-sensitive mutation in the cdc14 gene of the fission yeast Schizosaccharomyces pombe results in failure to form a septum. Cells become highly elongated and multinucleate as growth and nuclear division continue in the absence of cell division. This article describes the cloning of the cdc14 gene and the identification of its product, a protein of 240 amino acids, p28cdc14. A null allele of the cdc14 gene shows that the gene is essential for septum formation and completion of the cell-division cycle. Overexpression of the gene product, p28cdc14, causes cell-cycle arrest in late G2 before mitosis. Cells leaking past the block activate p34cdc2 kinase and show condensed chromosomes, but the normal rearrangements of the microtubules and microfilaments that are associated with the transition from interphase to mitosis do not occur. Overexpression of p28cdc14 in mutants, in which the timing of mitosis is altered, suggests that these effects may be mediated upstream of the mitotic inhibitor wee1. These data are consistent with the idea that p28cdc14 may play a role in both the initiation of mitosis and septum formation and, by doing so, be part of the mechanism that coordinates these two cell-cycle events.

Mechanisms of garnet growth in eclogites of the Zermatt-Saas Fee unit, Western Alps

THESIS ABSTRACT Garnets are one of the key metamorphic minerals used to study peak metamorphic conditions or crystallization ages. Equilibrium is typically assumed between the garnet and the matrix. This thesis attempts to understand garnet growth in the Zermatt-Saas Fee (ZSF) eclogites, and discusses consequences for Sm/Nd and Lu/Hf dating and the equilibrium assumption. All studied garnets from the ZSF eclogites are strongly zoned in Mn, Fe, Mg, and Ca. Methods based on chemical zoning patterns and on 3D spatial statistics indicate different growth mechanisms depending on the sample studied. Garnets from the Pfulwe area are grown in a system where surface kinetics likely dominated over intergranular diffusion kinetics. Garnets fram two other localities, Nuarsax and Lago di Cignana, seem to have grown in a system where intergranular diffusion kinetics were dominating over surface kinetics, at least during initial growth. Garnets reveal strong prograde REE+Y zoning. They contain narrow central peaks for Lu + Yb + Tm ± Er and at least one additional small peak towards the rim. The REE Sm + Eu + Gd + Tb ± Dy are depleted in the cores but show one prominent peak close to the rim. It is shown that these patterns cam be explained using a transient matrix diffusion model where REE uptake is limited by diffusion in the matrix surrounding the porphyroblast. The secondary peaks in the garnet profiles are interpreted to reflect thermally activated diffusion due to a temperature increase during prograde metamorphism. The model predicts anomalously low 176Lu/177Hf and 147Sm/144Nd ratios in garnets where growth rates are fast compared to diffusion of the REE, which decreases garnet isochron precisions. The sharp Lu zoning was further used to constrain maximum Lu volume diffusion rates in garnet. The modeled minimum pre-exponential diffusion coefficient which fits the measured central peak is in the order of Do = 5.7* 106 m2/s, taking an activation energy of 270 kJ/mol. The latter was chosen in agreement with experimentally determined values. This can be used to estimate a minimum closure temperature of around 630°C for the ZSF zone. Zoning of REE was combined with published Lu/Hf and Sm/Nd age information to redefine the prograde crystallization interval for Lago di Cignana UHP eclogites. Modeling revealed that a prograde growth interval in the order of 25 m.y. is needed to produce the measured spread in ages. RÉSUMÉ Le grenat est un minéral métamorphique clé pour déterminer les conditions du pic de métamorphisme ainsi que l'âge de cristallisation. L'équilibre entre le grenat et la matrice est requis. Cette étude a pour but de comprendre la croissance du grenat dans les éclogites de la zone de Zermatt-Saas Fee (ZSF) et d'examiner quelques conséquences sur les datations Sm/Nd et Lu/Hf. Tous les grenats des éclogites de ZSF étudiés sont fortement zonés en Mn, Fe, Mg et partiellement en Ca. Les différentes méthodes basées sur le modèle de zonation chimique ainsi que sur les statistiques de répartition spatiale en 3D indiquent un mécanisme de croissance différent en fonction de la localité d'échantillonnage. Les grenats provenant de la zone de Pfulwe ont probablement crû dans un système principalement dominé par la cinétique de surface au détriment de 1a cinétique de diffusion intergranulaire. Les grenats provenant de deux autres localités, Nuarsax et Lago di Cignana, semblent avoir cristallisé dans un système dominé par la diffusion intergranulaire, au moins durant les premiers stades de croissance. Les grenats montrent une forte zonation prograde en Terres Rares (REE) ainsi qu'en Y. Les profils présentent au coeur un pic étroit en Lu + Yb+ Tm ± Er et au moins un petit pic supplémentaire vers le bord. Les coeurs des grenats sont appauvris en Sm + Eu + Gd + Tb ± Dy, mais les bords sont marqués par un pic important de ces REE. Ces profils s'expliquent par un modèle de diffusion matricielle dans lequel l'apport en REE est limité par la diffusion dans la matrice environnant les porphyroblastes. Les pics secondaires en bordure de grain reflètent la diffusion activée par l'augmentation de la température lors du métamorphisme prograde. Ce modèle prédit des rapports 176Lu/177Hf et 147Sm/144Nd anormalement bas lorsque les taux de croissance sont plus rapides que la diffusion des REE, ce qui diminue la précision des isochrones impliquant le grenat. La zonation nette en Lu a permis de contraindre le maximum de diffusion volumique par une approche numérique. Le coefficient de diffusion minimum modélisé en adéquation avec les pics mesurés est de l'ordre de Do = 5.7*10-6 m2/s, en prenant une énergie d'activation ~270 kJ/mol déterminée expérimentalement. Ainsi, la température de clôture minimale est estimée aux alentours de 630°C pour la zone ZSF. Des nouvelles données de zonation de REE sont combinées aux âges obtenus avec les rapports Lu/Hf et Sm/Nd qui redéfissent l'intervalle de cristallisation prograde pour les éclogites UHP de Lago di Cignana. La modélisation permet d'attribuer au minimum un intervalle de croissance prograde de 25 Ma afin d'obtenir les âges préalablement mesurés. RESUME GRAND PUBLIC L'un des principaux buts du pétrologue .métamorphique est d'extraire des roches les informations sur l'évolution temporelle, thermique et barométrique qu'elles ont subi au cours de la formation d'une chaîne de montagne. Le grenat est l'un des minéraux clés dans une grande variété de roches métamorphiques. Il a fait l'objet de nombreuses études dans des terrains d'origines variées ou lors d'études expérimentales afin de comprendre ses domaines de stabilité, ses réactions et sa coexistence avec d'autres minéraux. Cela fait du grenat l'un des minéraux les plus attractifs pour la datation des roches. Cependant, lorsqu'on l'utilise pour la datation et/ou pour la géothermobarométrie, on suppose toujours que le grenat croît en équilibre avec les phases coexistantes de la matrice. Pourtant, la croissance d'un minéral est en général liée au processus de déséquilibre. Cette étude a pour but de comprendre comment croît le grenat dans les éclogites de Zermatt - Saas Fee et donc d'évaluer le degré de déséquilibre. Il s'agit aussi d'expliquer les différences d'âges obtenues grâce aux grenats dans les différentes localités de l'unité de Zermatt-Saas Fee. La principale question posée lors de l'étude des mécanismes de croissance du grenat est: Parmi les processus en jeu lors de la croissance du grenat (dissolution des anciens minéraux, transport des éléments vers le nouveau grenat, précipitation d'une nouvelle couche en surface du minéral), lequel est le plus lent et ainsi détermine le degré de déséquilibre? En effet, les grenats d'une des localités (Pfulwe) indiquent que le phénomène d'adhérence en surface est le plus lent, contrairement aux grenats des autres localités (Lago di Cignana, Nuarsax) dans lesquels ce sont les processus de transport qui sont les plus lents. Cela montre que les processus dominants sont variables, même dans des roches similaires de la même unité tectonique. Ceci implique que les processus doivent être déterminés individuellement pour chaque roche afin d'évaluer le degré de déséquilibre du grenat dans la roche. Tous les grenats analysés présentent au coeur une forte concentration de Terres Rares: Lu + Yb + Tm ± Er qui décroît vers le bord du grain. Inversement, les Terres Rares Sm + Eu + Gd + Tb ± Dy sont appauvries au coeur et se concentrent en bordure du grain. La modélisation révèle que ces profils sont-dus à des cinétiques lentes de transport des Terres Rares. De plus, les modèles prédisent des concentrations basses en éléments radiogéniques pères dans certaines roches, ce qui influence fortement sur la précision des âges obtenus par la méthode d'isochrone. Ceci signifie que les roches les plus adaptées pour les datations ne doivent contenir ni beaucoup de grenat ni de très gros cristaux, car dans ce cas, la compétition des éléments entre les cristaux limite à de faibles concentrations la quantité d'éléments pères dans chaque cristal.

Inhibition of the shade avoidance response by formation of non-DNA binding bHLH heterodimers.

In shade-intolerant plants such as Arabidopsis, a reduction in the red/far-red (R/FR) ratio, indicative of competition from other plants, triggers a suite of responses known as the shade avoidance syndrome (SAS). The phytochrome photoreceptors measure the R/FR ratio and control the SAS. The phytochrome-interacting factors 4 and 5 (PIF4 and PIF5) are stabilized in the shade and are required for a full SAS, whereas the related bHLH factor HFR1 (long hypocotyl in FR light) is transcriptionally induced by shade and inhibits this response. Here we show that HFR1 interacts with PIF4 and PIF5 and limits their capacity to induce the expression of shade marker genes and to promote elongation growth. HFR1 directly inhibits these PIFs by forming non-DNA-binding heterodimers with PIF4 and PIF5. Our data indicate that PIF4 and PIF5 promote SAS by directly binding to G-boxes present in the promoter of shade marker genes, but their action is limited later in the shade when HFR1 accumulates and forms non-DNA-binding heterodimers. This negative feedback loop is important to limit the response of plants to shade.

Plant Phototropic Growth.

Plants are photoautotrophic sessile organisms that use environmental cues to optimize multiple facets of growth and development. A classic example is phototropism - in shoots this is typically positive, leading to growth towards the light, while roots frequently show negative phototropism triggering growth away from the light. Shoot phototropism optimizes light capture of leaves in low light environments and hence increases photosynthetic productivity. Phototropins are plasma-membrane-associated UV-A/blue-light activated kinases that trigger phototropic growth. Light perception liberates their protein kinase domain from the inhibitory action of the amino-terminal photosensory portion of the photoreceptor. Following a series of still poorly understood events, phototropin activation leads to the formation of a gradient of the growth hormone auxin across the photo-stimulated stem. The greater auxin concentration on the shaded compared with the lit side of the stem enables growth reorientation towards the light. In this Minireview, we briefly summarize the signaling steps starting from photoreceptor activation until the establishment of a lateral auxin gradient, ultimately leading to phototropic growth in shoots.

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.