Hedgehog and Dpp signaling induce cadherin Cad86C expression in the morphogenetic furrow during Drosophila eye development

During Drosophila eye development, cell differentiation is preceded by the formation of a morphogenetic furrow, which progresses across the epithelium from posterior to anterior. Cells within the morphogenetic furrow are apically constricted and shortened along their apical-basal axis. However, how these cell shape changes and, thus, the progression of the morphogenetic furrow are controlled is not well understood. Here we show that cells simultaneously lacking Hedgehog and Dpp signal transduction fail to shorten and do not enter the morphogenetic furrow. Moreover, we have identified a gene, cadherin Cad86C, which is highly expressed in cells of the leading flank of the morphogenetic furrow. Ectopic activation of either the Hedgehog or Dpp signal transduction pathway results in elevated Cad86C expression. Conversely, simultaneous loss of both Hedgehog and Dpp signal transduction leads to decreased Cad86C expression. Finally, ectopic expression of Cad86C in either eye-antennal imaginal discs or wing imaginal discs results in apical constriction and shortening of cells. We conclude that Hedgehog and Dpp signaling promote the shortening of cells within the morphogenetic furrow. Induction of Cad86C expression might be one mechanism through which Hedgehog and Dpp promote these cell shape changes.     

Cholesterol in signal transduction

Membrane cholesterol impinges on signal transduction in several ways, which is highlighted in particular by the Hedgehog signaling pathway. In Hedgehog signaling, cholesterol is important for ligand biogenesis, as well as for signal transduction in receiving cells. Hedgehog ligands are post-translationally modified by cholesterol, and the Hedgehog receptor, Patched, is structurally similar to the Niemann-Pick C1 protein, which functions in intracellular lipid transport. Although the exact role of cholesterol in Hedgehog signal transduction remains elusive and is probably multifaceted, studies over the past year have implicated raft membrane subdomains, cholesterol transport and a link between protein and lipid trafficking in endocytic compartments.     

The EGF receptor and notch signaling pathways control the initiation of the morphogenetic furrow during Drosophila eye development

The onset of pattern formation in the developing Drosophila retina begins with the initiation of the morphogenetic furrow, the leading edge of a wave of retinal development that transforms a uniform epithelium, the eye imaginal disc into a near crystalline array of ommatidial elements. The initiation of this wave of morphogenesis is under the control of the secreted morphogens Hedgehog (Hh), Decapentaplegic (Dpp) and Wingless (Wg). We show that the Epidermal Growth Factor Receptor and Notch signaling cascades are crucial components that are also required to initiate retinal development. We also show that the initiation of the morphogenetic furrow is the sum of two genetically separable processes: (1) the 'birth' of pattern formation at the posterior margin of the eye imaginal disc; and (2) the subsequent 'reincarnation' of retinal development across the epithelium.     

Sprouty, an intracellular inhibitor of Ras signaling

Sprouty was identified in a genetic screen as an inhibitor of Drosophila EGF receptor signaling. The Egfr triggers cell recruitment in the eye, and sprouty- eyes have excess photoreceptors, cone cells, and pigment cells. Sprouty's function is, however, more widespread. We show that it also interacts genetically with the receptor tyrosine kinases Torso and Sevenless, and it was first discovered through its effect on FGF receptor signaling. In contrast to an earlier proposal that Sprouty is extracellular, we show by biochemical analysis that Sprouty is an intracellular protein, associated with the inner surface of the plasma membrane. Sprouty binds to two intracellular components of the Ras pathway, Drk and Gap1. Our results indicate that Sprouty is a widespread inhibitor of Ras pathway signal transduction.     

Activation of Smurf E3 Ligase Promoted by Smoothened Regulates Hedgehog Signaling through Targeting Patched Turnover

Hedgehog signaling plays conserved roles in controlling embryonic development; its dysregulation has been implicated in many human diseases including cancers. Hedgehog signaling has an unusual reception system consisting of two transmembrane proteins, Patched receptor and Smoothened signal transducer. Although activation of Smoothened and its downstream signal transduction have been intensively studied, less is known about how Patched receptor is regulated, and particularly how this regulation contributes to appropriate Hedgehog signal transduction. Here we identified a novel role of Smurf E3 ligase in regulating Hedgehog signaling by controlling Patched ubiquitination and turnover. Moreover, we showed that Smurf-mediated Patched ubiquitination depends on Smo activity in wing discs. Mechanistically, we found that Smo interacts with Smurf and promotes it to mediate Patched ubiquitination by targeting the K1261 site in Ptc. The further mathematic modeling analysis reveals that a bidirectional control of activation of Smo involving Smurf and Patched is important for signal-receiving cells to precisely interpret external signals, thereby maintaining Hedgehog signaling reliability. Finally, our data revealed an evolutionarily conserved role of Smurf proteins in controlling Hh signaling by targeting Ptc during development.     

Myosin II regulates complex cellular arrangement and epithelial architecture in Drosophila

Remodeling epithelia is a primary driver of morphogenesis. Here, we report a central role of myosin II in regulating several aspects of complex epithelial architecture in the Drosophila eye imaginal disc. The epithelial indentation of the morphogenetic furrow is established from a pattern of myosin II activation defined by the developmental signals Hedgehog and Decapentaplegic. More generally, patterned myosin activation can control diverse three-dimensional epithelial sculpting. We have developed a technique to image eye disc development in real time, and we show that myosin II also regulates higher-order organization of cells in the plane of the epithelium. This includes the clustering of cells into ommatidial units and their subsequent coordinated rotation. This later clustering function of myosin II depends on EGF receptor signaling. Our work implies that regulation of the actomyosin cytoskeleton can control morphogenesis by regulating both individual cell shapes and their complex two-dimensional arrangement within epithelia.     

Inhibition of EGF-Induced Signal Transduction by Microgravity Is Independent of EGF Receptor Redistribution in the Plasma Membrane of Human A431 Cells

Epidermal growth factor (EGF)-induced c-fos and c-jun expression is strongly suppressed in microgravity. We investigate here whether this is due to inhibition of processes occurring during the initiation of EGF-induced signal transduction. For this purpose, EGF-induced receptor clustering is used as a marker. The lateral distribution of EGF receptors is directly visualized at an ultrastructural level by the label-fracture method. Quantification of the receptor distributions shows that EGF-induced receptor redistribution is similar under normal and microgravity conditions. This suggests that microgravity influences EGF-induced signal transduction downstream of EGF binding and EGF receptor redistribution, but upstream of early gene expression in human A431 cells.     

Systems‐level interactions between insulin–EGF networks amplify mitogenic signaling

Crosstalk mechanisms have not been studied as thoroughly as individual signaling pathways. We exploit experimental and computational approaches to reveal how a concordant interplay between the insulin and epidermal growth factor (EGF) signaling networks can potentiate mitogenic signaling. In HEK293 cells, insulin is a poor activator of the Ras/ERK (extracellular signal‐regulated kinase) cascade, yet it enhances ERK activation by low EGF doses. We find that major crosstalk mechanisms that amplify ERK signaling are localized upstream of Ras and at the Ras/Raf level. Computational modeling unveils how critical network nodes, the adaptor proteins GAB1 and insulin receptor substrate (IRS), Src kinase, and phosphatase SHP2, convert insulin‐induced increase in the phosphatidylinositol‐3,4,5‐triphosphate (PIP₃) concentration into enhanced Ras/ERK activity. The model predicts and experiments confirm that insulin‐induced amplification of mitogenic signaling is abolished by disrupting PIP₃‐mediated positive feedback via GAB1 and IRS. We demonstrate that GAB1 behaves as a non‐linear amplifier of mitogenic responses and insulin endows EGF signaling with robustness to GAB1 suppression. Our results show the feasibility of using computational models to identify key target combinations and predict complex cellular responses to a mixture of external cues.     

Broad-complex, but not ecdysone receptor, is required for progression of the morphogenetic furrow in the Drosophila eye

The progression of the morphogenetic furrow in the developing Drosophila eye is an early metamorphic, ecdysteroid-dependent event. Although Ecdysone receptor-encoded nuclear receptor isoforms are the only known ecdysteroid receptors, we show that the Ecdysone receptor gene is not required for furrow function. DHR78, which encodes another candidate ecdysteroid receptor, is also not required. In contrast, zinc finger-containing isoforms encoded by the early ecdysone response gene Broad-complex regulate furrow progression and photoreceptor specification. br-encoded Broad-complex subfunctions are required for furrow progression and proper R8 specification, and are antagonized by other subfunctions of Broad-complex. There is a switch from Broad complex Z2 to Z1 zinc-finger isoform expression at the furrow which requires Z2 expression and responds to Hedgehog signals. These results suggest that a novel hormone transduction hierarchy involving an uncharacterized receptor operates in the eye disc.     

Loss of cilia causes embryonic lung hypoplasia,liver fibrosis,and cholestasis in the talpid3 ciliopathy mutant

Sonic hedgehog plays an essential role in maintaining hepatoblasts in a proliferative non-differentiating state during embryogenesis. Transduction of the Hedgehog signaling pathway is dependent on the presence of functional primary cilia and hepatoblasts, therefore, must require primary cilia for normal function. In congenital syndromes in which cilia are absent or non-functional (ciliopathies) hepatorenal fibrocystic disease is common and primarily characterized by ductal plate malformations which underlie the formation of liver cysts, as well as less commonly, by hepatic fibrosis, although a role for abnormal Hedgehog signal transduction has not been implicated in these phenotypes. We have examined liver, lung and rib development in the talpid³ chicken mutant, a ciliopathy model in which abnormal Hedgehog signaling is well characterized. We find that the talpid³ phenotype closely models that of human short-rib polydactyly syndromes which are caused by the loss of cilia, and exhibit hypoplastic lungs and liver failure. Through an analysis of liver and lung development in the talpid³ chicken, we propose that cilia in the liver are essential for the transduction of Hedgehog signaling during hepatic development. The talpid³ chicken represents a useful resource in furthering our understanding of the pathology of ciliopathies beyond the treatment of thoracic insufficiency as well as generating insights into the role Hedgehog signaling in hepatic development.     

Signal transduction of angiotensin II type 1 receptor through receptor tyrosine kinase

In cultured vascular smooth muscle cells, the angiotensin II (AngII) type-1 (AT(1)) receptor generates growth-promoting signals via the epidermal growth factor (EGF) receptor system. This 'transactivation' mechanism now appears to be utilized by a variety of G-protein-coupled receptors in many cells. The AngII-induced EGF receptor transactivation leads to activation of downstream signaling molecules including Ras, ERK, c-fos, Akt/protein kinase B, and p70 S6 kinase. We propose three possible mechanisms may be involved in the transactivation, (i) an upstream tyrosine kinase, (ii) reactive oxygen species, and (iii) a juxtacrine activation of the EGF receptor ligand. Whether the EGF receptor signal transduction induced by AngII plays an essential role in cardiovascular remodeling remains to be investigated.     

N-methyl-N'-nitro-N-nitrosoguanidine interferes with the epidermal growth factor receptor-mediated signaling pathway

Many environmental factors, such as ultraviolet (UV) and arsenic, can induce the clustering of cell surface receptors, including epidermal growth factor receptor (EGFR). This is accompanied by the phosphorylation of the receptors and the activation of ensuing cellular signal transduction pathways, which are implicated in the various cellular responses caused by the exposure to these factors. In this study, we have shown that N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), an alkylating agent, also induced the clustering of EGFR in human amnion FL cells, which was similar in morphology to that of epidermal growth factor treatment. However, MNNG treatment did not activate Ras, the downstream mediator in EGFR signaling pathway, as compared to EGF treatment. The autophosphorylation of tyrosine residues Y1068 and Y1173 at the intracellular domain of EGFR, which is related to Ras activation under EGF treatment, was also not observed by MNNG exposure. Interestingly, although MNNG did not affect the binding of EGF to EGFR, MNNG can interfere with EGF function. For instance, pre-incubating FL cells with MNNG inhibited the autophosphorylation of EGFR by EGF treatment, as well as the activation of Ras. In addition, the phosphorylation of Y845 on EGFR by EGF, which is mediated through c-Src or related kinases but not autophosphorylation, was also affected by MNNG. Therefore, MNNG may influence the tyrosine kinase activity as well as the phosphorylation of EGFR through its interaction with EGFR.     

Signaling,mitogenesis and the cytoskeleton: Where the action is

Stimulation of mitogenesis by the epidermal growth factor (EGF) operates through a pathway involving the receptor, the small G-protein Ras and protein kinases of the MAP kinase cascade. It is proposed that two of the critical steps of that pathway utilize localization of components to the plasma membrane where Ras is located: recruitment of the nucleotide exchange protein Sos to the phosphorylated EGF receptor via a complex with the SH2/SH3-containing protein Grb2 and recruitment of the protein kinase Raf to activated Ras. Moreover, it is then proposed that Raf associates with the cytoskeleton at the membrane as it is being activated. Other signaling elements, including class I receptor kinases, nonreceptor tyrosine kinases and tyrosine phosphatases, are known to function at specific cellular sites. These observations have led us to propose that localization of signaling components, and particularly sites at membrane-microfilament interfaces, play critical roles in cellular regulation.     

The EGF receptor: a nexus for trafficking and signaling

Ligand binding to the EGF receptor initiates both the activation of mitogenic signal transduction pathways plus trafficking events that relocalize the receptor on the cell surface and within intracellular compartments. The trafficking compartments include caveolae, clathrin-coated pits, and various endosome populations prior to receptor degradation in lysosomes. Evidence is presented that distinct signaling pathways are initiated from these different compartments. These include the Ras/MAP kinase cascade and the PLC-dependent hydrolysis of PI-4,5 P(2). Multiple tyrosine kinase substrates that facilitate EGF receptor trafficking between these various compartments, as well as the participation of phosphoinositides and Ras-like G proteins in the trafficking pathway are also described.     

Regulating mechanical tension at compartment boundaries in Drosophila

During animal development, cells with similar function and fate often stay together and sort out from cells with different fates. In Drosophila wing imaginal discs, cells of anterior and posterior fates are separated by a straight compartment boundary. Separation of anterior and posterior cells requires the homeodomain-containing protein Engrailed, which is expressed in posterior cells. Engrailed induces the expression of the short-range signaling molecule Hedgehog in posterior cells and confines Hedgehog signal transduction to anterior cells. Transduction of the Hedgehog signal in anterior cells is required for the separation of anterior and posterior cells. Previous work showed that this separation of cells involves a local increase in mechanical tension at cell junctions along the compartment boundary. However, how mechanical tension was locally increased along the compartment boundary remained unknown. A recent paper now shows that the difference in Hedgehog signal transduction between anterior and posterior cells is necessary and sufficient to increase mechanical tension. The local increase in mechanical tension biases junctional rearrangements during cell intercalations to maintain the straight shape of the compartment boundary. These data highlight how developmental signals can generate patterns of mechanical tension important for tissue organization.