Bud formation inHydra: Inhibition by an endogenous morphogen

Summary From crude extracts ofHydra tissue a substance has been purified which prevents or retards the asexual reproduction by budding. The molecular weight is in the range of 300 to 1000 daltons. Inhibition of bud formation can be observed with concentrations equivalent to the extract from one hydra per 4 ml, that is, to a more than 10,000-fold dilution of the initial crude extract of a hydra. The purified inhibitor is active at a concentration of less than 10^–8 M.Most of the inhibitor present inHydra is bound to cells. Within the cells the substance is mainly bound to particulate structures which sediment at 10,000 g. Its concentration is highest in the hypostomal region and decreases in the direction of the tentacles and peduncle. A second, lower, peak has been found in the basal disc. Treatment of the animals with a toxic agent (nitrogen mustard) which depletes the animal of interstitial cells, nematocytes and nematoblasts excludes the possibility that the inhibitor is present to any great extent in these cells. In conjunction with cell separation experiments by centrifugation of fixed cells in suspension, these results indicate that nerve cells are the most likely sites of storage of the inhibiting substance, although epithelial cells are not excluded as sources for the inhibitor.     

Wingless/Wnt signal transduction requires distinct initiation and amplification steps that both depend on Arrow/LRP

Members of the Wg/Wnt family provide key intercellular signals during embryonic development and in the maintenance of homeostatic processes, but critical aspects of their signal transduction pathways remain controversial. We have found that canonical Wg signaling in Drosophila involves distinct initiation and amplification steps, both of which require Arrow/LRP. Expressing a chimeric Frizzled2-Arrow protein in flies that lack endogenous Wg or Arrow showed that this construct functions as an activated Wg receptor but is deficient in signal amplification. In contrast, a chimeric Arrow protein containing the dimerization domain of Torso acted as a potent amplifier of Wg signaling but could not initiate Wg signaling on its own. The two chimeric proteins synergized, so that their co-expression largely reconstituted the signaling levels achieved by expressing Wg itself. The amplification function of Arrow/LRP appears to be particularly important for long-range signaling, and may reflect a general mechanism for potentiating signals in the shallow part of a morphogen gradient.     

Distance between AER and ZPA Is Defined by Feed-Forward Loop and Is Stabilized by their Feedback Loop in Vertebrate Limb Bud

In the development of organs, multiple morphogen sources are often involved, and interact with each other. For example, the apical ectodermal ridge (AER) and the zone of polarizing activity (ZPA) are major morphogen sources in the limb bud formation of vertebrates. Fgf expression in the AER and Shh expression in the ZPA are maintained by their positive feedback regulation mediated by diffusible molecules, FGF and SHH. A recent experimental observation suggests that the FGF-signal regulates the Shh expression in a feed-forward manner with activation and repression regulatory pathways. We study the coupled dynamics of Shh expression in the ZPA and Fgf expression in the AER, and the relationship of the relative position between AER and ZPA. We first show that with the feed-forward regulation only, the peak of ZPA activity can be formed distant from the AER as observed experimentally. Then, we clarify that the robustness of the ZPA spatial pattern to changes in system parameters is enhanced by adding the feedback regulation between the AER and the ZPA. Furthermore, sensitivity analysis shows that there exists the optimal feedback strength where the robustness is the most improved.     

Two distinct sites in sonic Hedgehog combine for heparan sulfate interactions and cell signaling functions

Hedgehog (Hh) proteins are morphogens that mediate many developmental processes. Hh signaling is significant for many aspects of embryonic development, whereas dysregulation of this pathway is associated with several types of cancer. Hh proteins require heparan sulfate proteoglycans (HSPGs) for their normal distribution and signaling activity. Here, we have used molecular modeling to examine the heparin-binding domain of sonic hedgehog (Shh). In biochemical and cell biological assays, the importance of specific residues of the putative heparin-binding domain for signaling was assessed. It was determined that key residues in human (h) Shh involved in heparin and HSPG syndecan-4 binding and biological activity included the well known cationic Cardin-Weintraub motif (lysines 32-38) but also a previously unidentified major role for lysine 178. The activity of Shh mutated in these residues was tested by quantitation of alkaline phosphatase activity in C3H10T1/2 cells differentiating into osteoblasts and hShh-inducible gene expression in PANC1 human pancreatic ductal adenocarcinoma cells. Mutated hShhs such as K37S/K38S, K178S, and particularly K37S/K38S/K178S that could not interact with heparin efficiently had reduced signaling activity compared with wild type hShh or a control mutation (K74S). In addition, the mutant hShh proteins supported reduced proliferation and invasion of PANC1 cells compared with control hShh proteins, following endogenous hShh depletion by RNAi knockdown. The data correlated with reduced Shh multimerization where the Lys-37/38 and/or Lys-178 mutations were examined. These studies provide a new insight into the functional roles of hShh interactions with HSPGs, which may allow targeting this aspect of hShh biology in, for example, pancreatic ductal adenocarcinoma.     

Spatiotemporally modulated Vestigial gradient by Wingless signaling adaptively regulates cell division for precise wing size control

In animal development, the growth of a tissue or organ is timely arrested when it reaches the stereotyped correct size. How this is robustly controlled remains poorly understood. The prevalent viewpoint, which is that morphogen gradients, due to their organizing roles in development, are directly responsible for growth arrest, cannot explain a number of observations. Recent findings from studies of the Drosophila wing have revealed that the interpretation of the Wingless gradient requires signaling-induced self-inhibition and that cell proliferation is controlled by graded vestigial expression. These findings highlight a growth control mechanism that involves Wingless regulated vestigial expression, but a question is whether they can quantitatively explain the observed preciseness and robustness of wing size control. Quantitative and systematic investigation into Wingless signaling using a mathematical model has elucidated two points. First, negative regulation of the Vestigial gradient by Wingless signaling makes vestigial expression precise and robust. Second, weak Wingless signaling in a primarily small wing pouch causes a short and steep Vestigial gradient, which stimulates more cell divisions and leads to a significant expansion of the wing pouch; however, strong Wingless signaling in a primarily large wing pouch causes a long and smooth Vestigial gradient, which stimulates fewer cell divisions and results in a slight expansion of the wing pouch. These results substantially decipher an inherent mechanism of tissue and organ size control. Our model explains, and is supported by, a number of experimental observations.     

BMP morphogen gradients in flies

Bone morphogenetic proteins (BMPs) act as morphogens to control patterning and growth in a variety of developing tissues in different species. How BMP morphogen gradients are established and interpreted in the target tissues has been extensively studied in Drosophila melanogaster. In Drosophila, Decapentaplegic (Dpp), a homologue of vertebrate BMP2/4, acts as a morphogen to control dorsal–ventral patterning of the early embryo and anterior–posterior patterning and growth of the wing imaginal disc. Despite intensive efforts over the last twenty years, how the Dpp morphogen gradient in the wing imaginal disc forms remains controversial, while gradient formation in the early embryo is well understood. In this review, we first focus on the current models of Dpp morphogen gradient formation in these two tissues, and then discuss new strategies using genome engineering and nanobodies to tackle open questions.