Identification of a Frizzled-like cysteine rich domain in the extracellular region of developmental receptor tyrosine kinases.

In Drosophila, members of the Frizzled family of tissue-polarity genes encode proteins that appear to function as cell-surface receptors for Wnts. The Frizzled genes belong to the seven transmembrane class of receptors (7TMR) and have on their extracellular region a cysteine-rich domain that has been implicated as the Wnt binding domain. This region has a characteristic spacing of ten cysteines, which has also been identified in FrzB (a secreted antagonist of Wnt signaling) and Smoothened (another 7TMR, which is involved in suppression of the hedgehog pathway). We have identified, using BLAST, sequence similarity between the cysteine-rich domain of Frizzled and several receptor tyrosine kinases, which have roles in development. These include the muscle-specific receptor tyrosine kinase (MuSK), the neuronal specific kinase (NSK2), and ROR1 and ROR2. At present, the ligands for these developmental tyrosine kinases are unknown. Our results suggest that Wnt-like ligands may bind to these developmental tyrosine kinases.     

The Wingless morphogen gradient is established by the cooperative action of Frizzled and Heparan Sulfate Proteoglycan receptors

We have examined the respective contribution of Heparan Sulfate Proteoglycans (HSPGs) and Frizzled (Fz) proteins in the establishment of the Wingless (Wg) morphogen gradient. From the analysis of mutant clones of sulfateless/N-deacetylase-sulphotransferase in the wing imaginal disc, we find that lack of Heparan Sulfate (HS) causes a dramatic reduction of both extracellular and intracellular Wg in receiving cells. Our studies, together with others [Kirkpatrick, C.A., Dimitroff, B.D., Rawson, J.M., Selleck, S.B., 2004. Spatial regulation of Wingless morphogen distribution and signalling by Dally-like protein. Dev. Cell (in press)], reveals that the Glypican molecule Dally-like Protein (Dlp) is associated with both negative and positive roles in Wg short- and long-range signaling, respectively. In addition, analyses of the two Fz proteins indicate that the Fz and DFz2 receptors, in addition to transducing the signal, modulate the slope of the Wg gradient by regulating the amount of extracellular Wg. Taken together, our analysis illustrates how the coordinated activities of HSPGs and Fz/DFz2 shape the Wg morphogen gradient.     

Dorsal-ventral signaling in limb development

In both Drosophila wings and vertebrate limbs, signaling between dorsal and ventral cells establishes an organizer that promotes limb formation. Significant progress has been made recently towards characterizing the signaling interactions that occur at the dorsal—ventral limb border. Studies of chicks have indicated that, as in Drosophila, this signaling process requires the participation of Fringe. Studies of Drosophila have indicated that Fringe functions by inhibiting the ability of Notch to be activated by one ligand, Serrate, while potentiating the ability of Notch to be activated by another ligand, Delta. Recent studies of both Drosophila and vertebrates have also shed new light on the signaling activity of the dorsal—ventral boundary limb organizer, and have highlighted how this organizer is maintained by feedback mechanisms with neighboring cells.     

Fast-tracking morphogen diffusion

The readout of morphogen concentrations has been proposed to be an essential mechanism allowing embryos to specify cell identities [Wolpert Trends Genet 12 (1996) 359], but theoretical and experimental results have led to conflicting ideas as to how useful concentration gradients can be established. In particular, it has been pointed out that some models of passive extracellular diffusion exhibit traveling waves of receptor saturation, inadequate for the establishment of positional information. Two alternative (but not mutually exclusive) models are proposed here, which are based on recent experimental results highlighting the roles of extracellular glycoproteins and morphogen oligomerization. In the first model, inspired from the interactions of Dally and Dally-like with Wingless and Decapentaplegic in the third-instar Drosophila wing disc, two morphogen populations are considered: one in a cell-membrane phase, and another one in an extracellular matrix phase, which does not interact with receptors; in the second model, inspired from biochemical studies of Sonic Hedgehog, morphogen oligomers are considered to diffuse freely without interacting with receptors. The existence of a dynamic sub-population of freely diffusing morphogen allows the system to establish a gradient of bound receptor that is suitable for the specification of positional information. Recent experimental results are discussed within the framework of these models, as well as further possible experiments. The role of Notum in the setup of the Wg gradient is also shown to be likely not to involve a gradient in Notum distribution, even though Notum is only expressed close to the source of Wg synthesis.     

Border of Notch activity establishes a boundary between the two dorsal appendage tube cell types

Boundaries establish and maintain separate populations of cells critical for organ formation. We show that Notch signaling establishes the boundary between two types of post-mitotic epithelial cells, the Rhomboid- and the Broad-positive cells. These cells will undergo morphogenetic movements to generate the two sides of a simple organ, the dorsal appendage tube of the Drosophila egg chamber. The boundary forms due to a difference in Notch levels in adjacent cells. The Notch expression pattern mimics the boundary; Notch levels are high in Rhomboid cells and low in Broad cells. Notch(-) mutant clones generate an ectopic boundary: ectopic Rhomboid cells arise in Notch(+) cells adjacent to the Notch(-) mutant cells but not further away from the clonal border. Pangolin, a component of the Wingless pathway, is required for Broad expression and for rhomboid repression. We further show that Broad represses rhomboid cell autonomously. Our data provide a foundation for understanding how a single row of Rhomboid cells arises adjacent to the Broad cells in the dorsal appendage primordia. Generating a boundary by the Notch pathway might constitute an evolutionarily conserved first step during organ formation in many tissues.     

On the role of glypicans in the process of morphogen gradient formation

Glypicans are cell surface molecules that influence signaling and gradient formation of secreted morphogens and growth factors. Several distinct functions have been ascribed to glypicans including acting as co-receptors for signaling proteins. Recent data show that glypicans are also necessary for morphogen propagation in the tissue. In the present study, a model describing the interaction of a morphogen with glypicans is formulated, analyzed and compared with measurements of the effect of glypican Dally-like (Dlp) overexpression on Wingless (Wg) morphogen signaling in Drosophila melanogaster wing imaginal discs. The model explains the opposing effect that Dlp overexpression has on Wg signaling in the distal and proximal regions of the disc and makes a number of quantitative predictions for further experiments. In particular, our model suggests that Dlp acts by allowing Wg to diffuse on cell surface while protecting it from loss and degradation, and that Dlp rather than acting as Wg co-receptor competes with receptors for morphogen binding.     

Wingless signaling and the control of cell shape in Drosophila wing imaginal discs

The control of cell morphology is important for shaping animals during development. Here we address the role of the Wnt/Wingless signal transduction pathway and two of its target genes, vestigial and shotgun (encoding E-cadherin), in controlling the columnar shape of Drosophila wing disc cells. We show that clones of cells mutant for arrow (encoding an essential component of the Wingless signal transduction pathway), vestigial or shotgun undergo profound cell shape changes and are extruded towards the basal side of the epithelium. Compartment-wide expression of a dominant-negative form of the Wingless transducer T-cell factor (TCF/Pangolin), or double-stranded RNA targeting vestigial or shotgun, leads to abnormally short cells throughout this region, indicating that these genes act cell autonomously to maintain normal columnar cell shape. Conversely, overexpression of Wingless, a constitutively-active form of the Wingless transducer β-catenin/Armadillo, or Vestigial, results in precocious cell elongation. Co-expression of Vestigial partially suppresses the abnormal cell shape induced by dominant-negative TCF. We conclude that Wingless signal transduction plays a cell-autonomous role in promoting and maintaining the columnar shape of wing disc cells. Furthermore, our data suggest that Wingless controls cell shape, in part, through maintaining vestigial expression.     

Homeogenetic inductive mechanism of segmentation in polychaete tail regeneration

Segmentation is a body-patterning strategy in which new segments are specified from a segment-addition zone containing uncommitted cells. However, the cell-recruitment process is poorly understood. Here we investigated in detail the segmentation in a polychaete annelid, Perinereis nuntia (Lophotrochozoa), in which new segments emerge at the boundary between the posterior end of the segmented region and the terminal pygidium. Cells at this border synchronously remodel their chromatin, enter the cell cycle, and undergo oriented cell division, before being added to new segments. wingless is expressed at the posterior edge of the pre-existing segment, abutted by hedgehog in the first row of the new segment. Overstimulation of Wingless signaling caused excess cells to enter the cell cycle, prolonging segmentation and widening the new segment. Thus, segment addition may occur by a homeogenetic mechanism, in which Wingless expressed in the differentiated segment coordinates the stepwise recruitment of undifferentiated cells from the segment/pygidium boundary.     

Drosophila Heparan Sulfate 6-O-Endosulfatase Sulf1 Facilitates Wingless (Wg) Protein Degradation

Heparan sulfate proteoglycans regulate various physiological and developmental processes through interactions with a number of protein ligands. Heparan sulfate (HS)-ligand binding depends on the amount and patterns of sulfate groups on HS, which are controlled by various HS sulfotransferases in the Golgi apparatus as well as extracellular 6-O-endosulfatases called “Sulfs.” Sulfs are a family of secreted molecules that specifically remove 6-O-sulfate groups within the highly sulfated regions on HS. Vertebrate Sulfs promote Wnt signaling, whereas the only Drosophila homologue of Sulfs, Sulf1, negatively regulates Wingless (Wg) signaling. To understand the molecular mechanism for the negative regulation of Wg signaling by Sulf1, we studied the effects of Sulf1 on HS-Wg interaction and Wg stability. Sulf1 overexpression strongly inhibited the binding of Wg to Dally, a potential target heparan sulfate proteoglycan of Sulf1. This effect of Drosophila Sulf1 on the HS-Wg interaction is similar to that of vertebrate Sulfs. Using in vitro, in vivo, and ex vivo systems, we show that Sulf1 reduces extracellular Wg protein levels, at least partly by facilitating Wg degradation. In addition, expression of human Sulf1 in the Drosophila wing disc lowers the levels of extracellular Wg protein, as observed for Drosophila Sulf1. Our study demonstrates that vertebrate and Drosophila Sulfs have an intrinsically similar activity and that the function of Sulfs in the fate of Wnt/Wg ligands is context-dependent.