In vivo imaging reveals different cellular functions for FGF and Dpp signaling in tracheal branching morphogenesis

In the developing tracheal system of Drosophila melanogaster, six major branches arise by guided cell migration from a sac-like structure. The chemoattractant Branchless/FGF (Bnl) appears to guide cell migration and is essential for the formation of all tracheal branches, while Decapentaplegic (Dpp) signaling is strictly required for the formation of a subset of branches, the dorsal and ventral branches. Using in vivo confocal video microscopy, we find that the two signaling systems affect different cellular functions required for branching morphogenesis. Bnl/FGF signaling affects the formation of dynamic filopodia, possibly controlling cytoskeletal activity and motility as such, and Dpp controls cellular functions allowing branch morphogenesis and outgrowth.     

Drosophila Dpp morphogen movement is independent of dynamin-mediated endocytosis but regulated by the glypican members of heparan sulfate proteoglycans

The Drosophila transforming growth factor beta (TGF-beta) homolog Decapentaplegic (Dpp) acts as a morphogen that forms a long-range concentration gradient to direct the anteroposterior patterning of the wing. Both planar transcytosis initiated by Dynamin-mediated endocytosis and extracellular diffusion have been proposed for Dpp movement across cells. In this work, we found that Dpp is mainly extracellular, and its extracellular gradient coincides with its activity gradient. We demonstrate that a blockage of endocytosis by the dynamin mutant shibire does not block Dpp movement but rather inhibits Dpp signal transduction, suggesting that endocytosis is not essential for Dpp movement but is involved in Dpp signaling. Furthermore, we show that Dpp fails to move across cells mutant for dally and dally-like (dly), two Drosophila glypican members of heparin sulfate proteoglycan (HSPG). Our results support a model in which Dpp moves along the cell surface by restricted extracellular diffusion involving the glypicans Dally and Dly.     

Dally regulates Dpp morphogen gradient formation by stabilizing Dpp on the cell surface

Decapentaplegic (Dpp), a Drosophila homologue of bone morphogenetic proteins, acts as a morphogen to regulate patterning along the anterior-posterior axis of the developing wing. Previous studies showed that Dally, a heparan sulfate proteoglycan, regulates both the distribution of Dpp morphogen and cellular responses to Dpp. However, the molecular mechanism by which Dally affects the Dpp morphogen gradient remains to be elucidated. Here, we characterized activity, stability, and gradient formation of a truncated form of Dpp (Dpp^ΔN), which lacks a short domain at the N-terminus essential for its interaction with Dally. Dpp^ΔN shows the same signaling activity and protein stability as wild-type Dpp in vitro but has a shorter half-life in vivo, suggesting that Dally stabilizes Dpp in the extracellular matrix. Furthermore, genetic interaction experiments revealed that Dally antagonizes the effect of Thickveins (Tkv; a Dpp type I receptor) on Dpp signaling. Given that Tkv can downregulate Dpp signaling by receptor-mediated endocytosis of Dpp, the ability of dally to antagonize tkv suggests that Dally inhibits this process. Based on these observations, we propose a model in which Dally regulates Dpp distribution and signaling by disrupting receptor-mediated internalization and degradation of the Dpp-receptor complex.     

Formation of the long range Dpp morphogen gradient

The TGF-β homolog Decapentaplegic (Dpp) acts as a secreted morphogen in the Drosophila wing disc, and spreads through the target tissue in order to form a long range concentration gradient. Despite extensive studies, the mechanism by which the Dpp gradient is formed remains controversial. Two opposing mechanisms have been proposed: receptor-mediated transcytosis (RMT) and restricted extracellular diffusion (RED). In these scenarios the receptor for Dpp plays different roles. In the RMT model it is essential for endocytosis, re-secretion, and thus transport of Dpp, whereas in the RED model it merely modulates Dpp distribution by binding it at the cell surface for internalization and subsequent degradation. Here we analyzed the effect of receptor mutant clones on the Dpp profile in quantitative mathematical models representing transport by either RMT or RED. We then, using novel genetic tools, experimentally monitored the actual Dpp gradient in wing discs containing receptor gain-of-function and loss-of-function clones. Gain-of-function clones reveal that Dpp binds in vivo strongly to the type I receptor Thick veins, but not to the type II receptor Punt. Importantly, results with the loss-of-function clones then refute the RMT model for Dpp gradient formation, while supporting the RED model in which the majority of Dpp is not bound to Thick veins. Together our results show that receptor-mediated transcytosis cannot account for Dpp gradient formation, and support restricted extracellular diffusion as the main mechanism for Dpp dispersal. The properties of this mechanism, in which only a minority of Dpp is receptor-bound, may facilitate long-range distribution.     

Direct competition between Brinker and Drosophila Mad in Dpp target gene transcription

Brinker is a nuclear protein that antagonizes Dpp signalling in Drosophila. Its expression is negatively regulated by Dpp. Here, we show that Brinker represses Ultrabithorax (Ubx) in the embryonic midgut, a HOX gene that activates, and responds to, the localized expression of Dpp during endoderm induction. We find that the functional target for Brinker repression coincides with the Dpp response sequence in the Ubx midgut enhancer, namely a tandem of binding sites for the Dpp effector Mad. We show that Brinker efficiently competes with Mad in vitro, preventing the latter from binding to these sites. Brinker also competes with activated Mad in vivo, blocking the stimulation of the Ubx enhancer in response to simultaneous Dpp signalling. These results indicate how Brinker acts as a dominant repressor of Dpp target genes, and explain why Brinker is a potent antagonist of Dpp.     

Repression of dpp targets by binding of brinker to mad sites

Signaling by decapentaplegic (Dpp), a Drosophila member of the transforming growth factor (TGF) beta superfamily of growth factors, has recently been shown to activate targets such as vestigial (vg) indirectly through negative regulation of brinker (brk). Here we show that the Brk protein functions as a repressor by binding to Dpp response elements. The Brk DNA binding activity was localized to an amino-terminal region containing a putative homeodomain. Brk bound to a Dpp response element of the Ultrabithorax (Ubx) midgut enhancer at a sequence that overlaps a binding site for the Smad protein, Mothers Against Dpp (Mad). Furthermore, Brk was able to compete with Mad for occupancy of this binding site. This recognition of overlapping binding sites provides a potential explanation for why the G/C-rich Mad binding site consensus differs the Smad3/Smad4 binding site consensus. We also found that the Dpp response element from Ubx was more sensitive than the vg quadrant enhancer to repression by Brk. This difference correlates with short-range activation of Ubx by Dpp in the visceral mesoderm, whereas vg exhibits a long-range response to Dpp in the wing imaginal disc, indicating that Brk binding sites may play a critical role in limiting thresholds for activation by Dpp. Finally, we provide evidence that Brk is capable of functioning as an active repressor. Thus, whereas Brk and Mad compete for regulation of Ubx and vg, Brk may regulate other Dpp targets without direct involvement of Mad.     

Incorporating chemical signalling factors into cell-based models of growing epithelial tissues

In this paper we present a comprehensive computational framework within which the effects of chemical signalling factors on growing epithelial tissues can be studied. The method incorporates a vertex-based cell model, in conjunction with a solver for the governing chemical equations. The vertex model provides a natural mesh for the finite element method (FEM), with node movements determined by force laws. The arbitrary Lagrangian-Eulerian formulation is adopted to account for domain movement between iterations. The effects of cell proliferation and junctional rearrangements on the mesh are also examined. By implementing refinements of the mesh we show that the finite element (FE) approximation converges towards an accurate numerical solution. The potential utility of the system is demonstrated in the context of Decapentaplegic (Dpp), a morphogen which plays a crucial role in development of the Drosophila imaginal wing disc. Despite the presence of a Dpp gradient, growth is uniform across the wing disc. We make the growth rate of cells dependent on Dpp concentration and show that the number of proliferation events increases in regions of high concentration. This allows hypotheses regarding mechanisms of growth control to be rigorously tested. The method we describe may be adapted to a range of potential application areas, and to other cell-based models with designated node movements, to accurately probe the role of morphogens in epithelial tissues.     

Dynamic decapentaplegic signaling regulates patterning and adhesion in the Drosophila pupal retina

The correct organization of cells within an epithelium is essential for proper tissue and organ morphogenesis. The role of Decapentaplegic/Bone morphogenetic protein (Dpp/BMP) signaling in cellular morphogenesis during epithelial development is poorly understood. In this paper, we used the developing Drosophila pupal retina--looking specifically at the reorganization of glial-like support cells that lie between the retinal ommatidia--to better understand the role of Dpp signaling during epithelial patterning. Our results indicate that Dpp pathway activity is tightly regulated across time in the pupal retina and that epithelial cells in this tissue require Dpp signaling to achieve their correct shape and position within the ommatidial hexagon. These results point to the Dpp pathway as a third component and functional link between two adhesion systems, Hibris-Roughest and DE-cadherin. A balanced interplay between these three systems is essential for epithelial patterning during morphogenesis of the pupal retina. Importantly, we identify a similar functional connection between Dpp activity and DE-cadherin and Rho1 during cell fate determination in the wing, suggesting a broader link between Dpp function and junctional integrity during epithelial development.     

Establishment of medial fates along the proximodistal axis of the Drosophila leg through direct activation of dachshund by Distalless

The proximodistal (PD) axis of the Drosophila leg is thought to be established by the combined gradients of two secreted morphogens, Wingless (Wg) and Decapentaplegic (Dpp). According to this model, high [Wg+Dpp] activates Distalless (Dll) and represses dachshund (dac) in the distal cells of the leg disc, while intermediate [Wg+Dpp] activates dac in medial tissue. To test this model we identified and characterized a dac cis-regulatory element (dac RE) that recapitulates dac's medial expression domain during leg development. Counter to the gradient model, we find that Wg and Dpp do not act in a graded manner to activate RE. Instead, dac RE is activated directly by Dll and repressed distally by a combination of factors, including the homeodomain protein Bar. Thus, medial leg fates are established via a regulatory cascade in which Wg+Dpp activate Dll and then Dll directly activates dac, with Wg+Dpp as less critical, permissive inputs.     

A characterization of the effects of Dpp signaling on cell growth and proliferation in the Drosophila wing

Cell proliferation and patterning must be coordinated for the development of properly proportioned organs. If the same molecules were to control both processes, such coordination would be ensured. Here we address this possibility in the Drosophila wing using the Dpp signaling pathway. Previous studies have shown that Dpp forms a gradient along the AP axis that patterns the wing, that Dpp receptors are autonomously required for wing cell proliferation, and that ectopic expression of either Dpp or an activated Dpp receptor, Tkv(Q253D), causes overgrowth. We extend these findings with a detailed analysis of the effects of Dpp signaling on wing cell growth and proliferation. Increasing Dpp signaling by expressing Tkv(Q253D) accelerated wing cell growth and cell cycle progression in a coordinate and cell-autonomous manner. Conversely, autonomously inhibiting Dpp signaling using a pathway specific inhibitor, Dad, or a mutation in tkv, slowed wing cell growth and division, also in a coordinate fashion. Stimulation of cell cycle progression by Tkv(Q253D) was blocked by the cell cycle inhibitor RBF, and required normal activity of the growth effector, PI3K. Among the known Dpp targets, vestigial was the only one tested that was required for Tkv(Q253D)-induced growth. The growth response to altering Dpp signaling varied regionally and temporally in the wing disc, indicating that other patterned factors modify the response.     

Facilitated transport of a Dpp/Scw heterodimer by Sog/Tsg leads to robust patterning of the Drosophila blastoderm embryo

Patterning the dorsal surface of the Drosophila blastoderm embryo requires Decapentaplegic (Dpp) and Screw (Scw), two BMP family members. Signaling by these ligands is regulated at the extracellular level by the BMP binding proteins Sog and Tsg. We demonstrate that Tsg and Sog play essential roles in transporting Dpp to the dorsal-most cells. Furthermore, we provide biochemical and genetic evidence that a heterodimer of Dpp and Scw, but not the Dpp homodimer, is the primary transported ligand and that the heterodimer signals synergistically through the two type I BMP receptors Tkv and Sax. We propose that the use of broadly distributed Dpp homodimers and spatially restricted Dpp/Scw heterodimers produces the biphasic signal that is responsible for specifying the two dorsal tissue types. Finally, we demonstrate mathematically that heterodimer levels can be less sensitive to changes in gene dosage than homodimers, thereby providing further selective advantage for using heterodimers as morphogens.     

A novel function of Drosophila eIF4A as a negative regulator of Dpp/BMP signalling that mediates SMAD degradation

Signalling by the TGF-beta superfamily member and BMP orthologue Decapentaplegic (Dpp) is crucial for multiple developmental programmes and has to be tightly regulated. Here, we demonstrate that the Drosophila Dpp pathway is negatively regulated by eukaryotic translation initiation factor 4A (eIF4A), which mediates activation-dependent degradation of the Dpp signalling components Mad and Medea. eIF4A mutants exhibit increased Dpp signalling and accumulation of Mad and phospho-Mad. Overexpression of eIF4A decreases Dpp signalling and causes loss of Mad and phospho-Mad. Furthermore, eIF4A physically associates with Mad and Medea, and promotes their degradation following activation of Dpp signalling in a translation-independent manner. Finally, we show that eIF4A acts synergistically with, but independently of, the ubiquitin ligase DSmurf, indicating that a dual system controls SMAD degradation. Thus, in addition to being an obligatory component of the cap-dependent translation initiation complex, eIF4A has a novel function as a specific inhibitor of Dpp signalling that mediates the degradation of SMAD homologues.