RacGap50C negatively regulates wingless pathway activity during Drosophila embryonic development

The Wingless (Wg)/Wnt signal transduction pathway directs a variety of cell fate decisions in developing animal embryos. Despite the identification of many Wg pathway components to date, it is still not clear how these elements work together to generate cellular identities. In the ventral epidermis of Drosophila embryos, Wg specifies cells to secrete a characteristic pattern of denticles and naked cuticle that decorate the larval cuticle at the end of embryonic development. We have used the Drosophila ventral epidermis as our assay system in a series of genetic screens to identify new components involved in Wg signaling. Two mutant lines that modify wg-mediated epidermal patterning represent the first loss-of-function mutations in the RacGap50C gene. These mutations on their own cause increased stabilization of Armadillo and cuticle pattern disruptions that include replacement of ventral denticles with naked cuticle, which suggests that the mutant embryos suffer from ectopic Wg pathway activation. In addition, RacGap50C mutations interact genetically with naked cuticle and Axin, known negative regulators of the Wg pathway. These phenotypes suggest that the RacGap50C gene product participates in the negative regulation of Wg pathway activity.     

Hox-controlled reorganisation of intrasegmental patterning cues underlies Drosophila posterior spiracle organogenesis

Hox proteins provide axial positional information and control segment morphology in development and evolution. Yet how they specify morphological traits that confer segment identity and how axial positional information interferes with intrasegmental patterning cues during organogenesis remain poorly understood. We have investigated the control of Drosophila posterior spiracle morphogenesis, a segment-specific structure that forms under Abdominal-B (AbdB) Hox control in the eighth abdominal segment (A8). We show that the Hedgehog (Hh), Wingless (Wg) and Epidermal Growth Factor Receptor (Egfr) pathways provide specific inputs for posterior spiracle morphogenesis and act in a genetic network made of multiple and rapidly evolving Hox/signalling interplays. A major function of AbdB during posterior spiracle organogenesis is to reset A8 intrasegmental patterning cues, first by reshaping wg and rhomboid expression patterns, then by reallocating the Hh signal and later by initiating de novo expression of the posterior compartment gene engrailed in anterior compartment cells. These changes in expression patterns confer axial specificity to otherwise reiteratively used segmental patterning cues, linking intrasegmental polarity and acquisition of segment identity.     

Patterning of Drosophila leg sensory organs through combinatorial signaling by hedgehog, decapentaplegic and wingless.

During development, global patterning events initiate signal transduction cascades which gradually establish an array of individual cell fates. Many of the genes which pattern Drosophila are expressed throughout development and specify diverse cell types by creating unique local environments which establish the expression of locally acting genes. This process is exemplified by the patterning of leg microchaete rows. hairy (h) is expressed in a spatially restricted manner in the leg imaginal disc and functions to position adult leg bristle rows by negatively regulating the proneural gene achaete, which specifies sensory cell fates. While much is known about the events that partition the leg imaginal disc and about sensory cell differentiation, the mechanisms that refine early patterning events to the level of individual cell fate specification are not well understood. We have investigated the regulation of h expression along the dorsal/ventral (D/V) axis of the leg adjacent to the anterior/posterior (A/P) compartment boundary and have found that it requires input from both D/V and A/P patterning mechanisms. Expression of the D/V axis h stripe (D/V-h) is controlled by dorsal- and ventral-specific enhancer elements which are targets of Decapentaplegic (Dpp) and Wingless (Wg) signaling, respectively, but which are also dependent on Hedgehog (Hh) signaling for activation. D/V-h expression is lost in smoothened mutant clones and is specifically activated by exogenously supplied Cubitus interruptus (Ci). D/V-h expression is also lost in clones deficient for Dpp and Wg signaling, but ectopic activation of D/V-h by Dpp and Wg is limited to the A/P compartment boundary where endogenous levels of full-length Ci are high. We propose that D/V-h expression is regulated in a non-linear pathway in which Ci plays a dual role. In addition to serving as an upstream activator of Dpp and Wg, Ci acts combinatorially with them to activate D/V-h expression.     

Three Drosophila EXT genes shape morphogen gradients through synthesis of heparan sulfate proteoglycans

The signaling molecules Hedgehog (Hh), Decapentaplegic (Dpp) and Wingless (Wg) function as morphogens and organize wing patterning in Drosophila. In the screen for mutations that alter the morphogen activity, we identified novel mutants of two Drosophila genes, sister of tout-velu (sotv) and brother of tout-velu (botv), and new alleles of tout-velu (ttv). The encoded proteins of these genes belong to an EXT family of proteins that have or are closely related to glycosyltransferase activities required for biosynthesis of heparan sulfate proteoglycans (HSPGs). Mutation in any of these genes impaired biosynthesis of HSPGs in vivo, indicating that, despite their structural similarity, they are not redundant in the HSPG biosynthesis. Protein levels and signaling activities of Hh, Dpp and Wg were reduced in the cells mutant for any of these EXT genes to a various degree, Wg signaling being the least sensitive. Moreover, all three morphogens were accumulated in the front of EXT mutant cells, suggesting that these morphogens require HSPGs to move efficiently. In contrast to previous reports that ttv is involved exclusively in Hh signaling, we found that ttv mutations also affected Dpp and Wg. These data led us to conclude that each of three EXT genes studied contribute to Hh, Dpp and Wg morphogen signaling. We propose that HSPGs facilitate the spreading of morphogens and therefore, function to generate morphogen concentration gradients.     

Cellular trafficking of the glypican Dally-like is required for full-strength Hedgehog signaling and wingless transcytosis

Hedgehog (Hh) and Wingless (Wg) morphogens specify cell fate in a concentration-dependent manner in the Drosophila wing imaginal disc. Proteoglycans, components of the extracellular matrix, are involved in Hh and Wg stability, spreading, and reception. In this study, we demonstrate that the glycosyl-phosphatidyl-inositol (GPI) anchor of the glypican Dally-like (Dlp) is required for its apical internalization and its subsequent targeting to the basolateral compartment of the epithelium. Dlp endocytosis from the apical surface of Hh-receiving cells catalyzes the internalization of Hh bound to its receptor Patched (Ptc). The cointernalization of Dlp with the Hh/Ptc complex is dynamin dependent and necessary for full-strength Hh signaling. We also demonstrate that Wg is secreted apically in the disc epithelium and that apicobasal trafficking of Dlp allows Wg transcytosis to favor Wg spreading along the basolateral compartment. Thus, Dlp endocytosis is a common regulatory mechanism of both Hh and Wg morphogen action.     

Mutual antagonism between signals secreted by adjacent wingless and engrailed cells leads to specification of complementary regions of the Drosophila parasegment.

Specialized groups of cells known as organizers govern the establishment of cell type diversity across cellular fields. Segmental patterning within the Drosophila embryonic epidermis is one paradigm for organizer function. Here cells differentiate into smooth cuticle or distinct denticle types. At parasegment boundaries, cells expressing Wingless confront cells co-expressing Engrailed and Hedgehog. While Wingless is essential for smooth cell fates, the signals that establish denticle diversity are unknown. We show that wg mutants have residual mirror-symmetric pattern that is due to an Engrailed-dependent signal specifying anterior denticle fates. The Engrailed-dependent signal acts unidirectionally and Wg activity imposes this asymmetry. Reciprocally, the Engrailed/Hedgehog interface imposes asymmetry on Wg signaling. Thus, a bipartite organizer, with each signal acting essentially unidirectionally, specifies segmental pattern.     

Non-equivalent roles of Drosophila Frizzled and Dfrizzled2 in embryonic wingless signal transduction

The highly conserved Wnt family of growth factors is essential for generating embryonic pattern in many animal species [1]. In the fruit fly Drosophila, most Wnt-mediated patterning is performed by a single family member, Wingless (Wg), acting through its receptors Frizzled (Fz) and DFrizzled2 (Dfz2). In the ventral embryonic epidermis, Wg signaling generates two different cell-fate decisions: the production of diverse denticle types and the specification of naked cuticle separating the denticle belts. Mutant alleles of wg disrupt these cellular decisions separately [2], suggesting that some aspect of ligand-receptor affinity influences cell-fate decisions, or that different receptor complexes mediate the distinct cellular responses. Here, we report that overexpression of Dfz2, but not Fz, rescues the mutant phenotype of wgPE2, an allele that produces denticle diversity but no naked cuticle. Fz was able to substitute for Dfz2 only under conditions where the Wg ligand was present in excess. The wgPE2 mutant phenotype was also sensitive to the dosage of glycosaminoglycans, suggesting that the mutant ligand is excluded from the receptor complex when proteoglycans are present. We conclude that wild-type Wg signaling requires efficient interaction between ligand and the Dfz2-proteoglycan receptor complex to promote the naked cuticle cell fate.     

Notch and Wingless modulate the response of cells to Hedgehog signalling in the Drosophila wing

During Drosophila wing development, Hedgehog (Hh) signalling is required to pattern the imaginal disc epithelium along the anterior-posterior (AP) axis. The Notch (N) and Wingless (Wg) signalling pathways organise the dorsal-ventral (DV) axis, including patterning along the presumptive wing margin. Here, we describe a functional hierarchy of these signalling pathways that highlights the importance of competing influences of Hh, N, and Wg in establishing gene expression domains. Investigation of the modulation of Hh target gene expression along the DV axis of the wing disc revealed that collier/knot (col/kn), patched (ptc), and decapentaplegic (dpp) are repressed at the DV boundary by N signalling. Attenuation of Hh signalling activity caused by loss of fused function results in a striking down-regulation of col, ptc, and engrailed (en) symmetrically about the DV boundary. We show that this down-regulation depends on activity of the canonical Wg signalling pathway. We propose that modulation of the response of cells to Hh along the future proximodistal (PD) axis is necessary for generation of the correctly patterned three-dimensional adult wing. Our findings suggest a paradigm of repression of the Hh response by N and/or Wnt signalling that may be applicable to signal integration in vertebrate appendages.     

Hedgehog signaling induces arterial endothelial cell formation by repressing venous cell fate

In vertebrate embryos, the dorsal aorta and the posterior cardinal vein form in the trunk to comprise the original circulatory loop. Previous studies implicate Hedgehog (Hh) signaling in the development of the dorsal aorta. However, the mechanism controlling specification of artery versus vein remains unclear. Here, we investigated the cell-autonomous mechanism of Hh signaling in angioblasts (endothelial progenitor cells) during arterial-venous specification utilizing zebrafish mutations in Smoothened (Smo), a G protein-coupled receptor essential for Hh signaling. smo mutants exhibit an absence of the dorsal aorta accompanied by a reciprocal expansion of the posterior cardinal vein. The increased number of venous cells is equivalent to the loss of arterial cells in embryos with loss of Smo function. Activation of Hh signaling expands the arterial cell population at the expense of venous cell fate. Time-lapse imaging reveals two sequential waves of migrating progenitor cells that contribute to the dorsal aorta and the posterior cardinal vein, respectively. Angioblasts deficient in Hh signaling fail to contribute to the arterial wave; instead, they all migrate medially as a single population to form the venous wave. Cell transplantation analyses demonstrate that Smo plays a cell-autonomous role in specifying angioblasts to become arterial cells, and Hh signaling-depleted angioblasts differentiate into venous cells instead. Collectively, these studies suggest that arterial endothelial cells are specified and formed via repressing venous cell fate at the lateral plate mesoderm by Hh signaling during vasculogenesis.     

Distinct signals generate repeating striped pattern in the embryonic parasegment

How repeating striped patterns arise across cellular fields is unclear. To address this we examined the repeating pattern of Stripe (Sr) expression across the parasegment (PS) in Drosophila. This pattern is generated in two steps. First, the ligands Hedgehog (Hh) and Wingless (Wg) subdivide the PS into smaller territories. Second, the ligands Hh, Spitz (Spi), and Wg each emanate from a specific territory and induce Sr expression in an adjacent territory. We also show that the width of Sr expression is determined by signaling strength. Finally, an enhancer trap in the sr gene detects the response to Spi and Wg, but not to Hh, implying the existence of separable control elements in the sr gene. Thus, a distinct inductive event is used to initiate each element of the repeating striped pattern.