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.     

Read-Out of Dynamic Morphogen Gradients on Growing Domains

Quantitative data from the Drosophila wing imaginal disc reveals that the amplitude of the Decapentaplegic (Dpp) morphogen gradient increases continuously. It is an open question how cells can determine their relative position within a domain based on a continuously increasing gradient. Here we show that pre-steady state diffusion-based dispersal of morphogens results in a zone within the growing domain where the concentration remains constant over the patterning period. The position of the zone that is predicted based on quantitative data for the Dpp morphogen corresponds to where the Dpp-dependent gene expression boundaries of spalt (sal) and daughters against dpp (dad) emerge. The model also suggests that genes that are scaling and are expressed at lateral positions are either under the control of a different read-out mechanism or under the control of a different morphogen. The patterning mechanism explains the extraordinary robustness that is observed for variations in Dpp production, and offers an explanation for the dual role of Dpp in controlling patterning and growth. Pre-steady-state dynamics are pervasive in morphogen-controlled systems, thus making this a probable general mechanism for the scaled read-out of morphogen gradients in growing developmental systems.     

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.     

Evidence for Wg-independent tergite boundary formation in the millipede Glomeris marginata

The correlation between dorsal and ventral segmental units in diplopod myriapods is complex and disputed. Recent results with engrailed (en), hedgehog (hh), wingless (wg), and cubitus-interruptus (ci) have shown that the dorsal segments are patterned differently from the ventral segments. Ventrally, gene expression is compatible with the classical autoregulatory loop known from Drosophila to specify the parasegment boundary. In the dorsal segments, however, this Wg/Hh autoregulatory loop cannot be present because the observed gene expression patterns argue against the involvement of Wg signalling. In this paper, we present further evidence against an involvement of Wg signalling in dorsal segmentation and propose a hypothesis about how dorsal segmental boundaries may be controlled in a wg-independent way. We find that (1) the Notum gene, a modulator of the Wg gradient in Drosophila, is not expressed in the dorsal segments. (2) The H15/midline gene, a repressor of Wg action in Drosophila, is not expressed in the dorsal segments, except for future heart tissue. (3) The patched (ptc) gene, which encodes a Hh receptor, is strongly expressed in the dorsal segments, which is incompatible with Wg-Hh autoregulation. The available data suggest that anterior-posterior (AP) boundary formation in dorsal segments could instead rely on Dpp signalling rather than Wg signalling. We present a hypothesis that relies on Hh-mediated activation of Dpp signalling and optomotor-blind (omb) expression to establish the dorsal AP boundary (the future tergite boundary). The proposed mechanism is similar to the mechanism used to establish the AP boundary in Drosophila wings and ventral pleura.     

Expanded and fat regulate growth and differentiation in the Drosophila eye through multiple signaling pathways

Mutations in the expanded gene act as hyperplastic tumor suppressors, interfere with cell competition and elevate Dpp signaling. Unlike Dpp overexpression, ex causes few patterning defects. Our data suggest that patterning effects are partly masked by antagonistic roles of other signaling pathways that are also activated. ex causes proliferation of cells in the posterior eye disc that are normally postmitotic. ex mutations elevate Wg signaling, but Dpp signaling antagonizes patterning effects of Wg. By contrast, if Dpp signaling is blocked in ex mutant cells, the elevated Wg signaling preserves an immature developmental state and prevents retinal differentiation. An effect of ex mutations on vesicle transport is suggested by evidence for altered sterol distribution. Mutations in ft show effects on proliferation, Wg signaling and sterols very similar to those of ex mutations. During disc growth, ex was largely epistatic to ft, and the Warts pathway mutation hippo largely epistatic to ex. Our data suggest that ft and ex act partially through the Warts pathway.     

Morphogenetic apoptosis: a mechanism for correcting discontinuities in morphogen gradients

Smooth gradients of the morphogens Hh, Dpp, and Wg are required for proper development of Drosophila imaginal discs. Here, it is reported that, when a discontinuity is generated between two adjacent cells in the reception of either the Dpp or Wg signal, then cells on either side of the discontinuity boundary undergo apoptosis by activating the c-Jun N-terminal Kinase (JNK) pathway. Furthermore, in the medial region of the wing imaginal disc, the JNK pathway is also activated if cells do not receive the proper levels of Dpp and Hh signals. These observations suggest that cells within a developing field have the ability to access their spatial positions by comparing the level of morphogen signal they receive with that of their neighbors. This phenomenon is likely related to the process of cell competition, and we suggest that it is an evolutionarily important mechanism that helps prevent abnormal tissue specification and growth during development.     

Genetic interaction of Lobe with its modifiers in dorsoventral patterning and growth of the Drosophila eye

Dorsoventral (DV) patterning is essential for growth of the Drosophila eye. Recent studies suggest that ventral is the default state of the early eye, which depends on Lobe (L) function, and that the dorsal fate is established later by the expression of the dorsal selector gene pannier (pnr). However, the mechanisms of regulatory interactions between L and dorsal genes are not well understood. For studying the mechanisms of DV patterning in the early eye disc, we performed a dominant modifier screen to identify additional genes that interact with L. The criterion of the dominant interaction was either enhancement or suppression of the L ventral eye loss phenotype. We identified 48 modifiers that correspond to 16 genes, which include fringe (fng), a gene involved in ventral eye patterning, and members of both Hedgehog (Hh) and Decapentaplegic (Dpp) signaling pathways, which promote L function in the ventral eye. Interestingly, 29% of the modifiers (6 enhancers and 9 suppressors) identified either are known to interact genetically with pnr or are members of the Wingless (Wg) pathway, which acts downstream from pnr. The detailed analysis of genetic interactions revealed that pnr and L mutually antagonize each other during second instar of larval development to restrict their functional domains in the eye. This time window coincides with the emergence of pnr expression in the eye. Our results suggest that L function is regulated by multiple signaling pathways and that the mutual antagonism between L and dorsal genes is crucial for balanced eye growth.     

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.     

Distinct and collaborative roles of Drosophila EXT family proteins in morphogen signalling and gradient formation

Heparan sulfate proteoglycans (HSPG) have been implicated in regulating the signalling activities of secreted morphogen molecules including Wingless (Wg), Hedgehog (Hh) and Decapentaplegic (Dpp). HSPG consists of a protein core to which heparan sulfate (HS) glycosaminoglycan (GAG) chains are attached. The formation of HS GAG chains is catalyzed by glycosyltransferases encoded by members of the EXT family of putative tumor suppressors linked to hereditary multiple exostoses. Previous studies in Drosophila demonstrated that tout-velu (ttv), the Drosophila EXT1, is required for Hh movement. However, the functions of other EXT family members are unknown. We have identified and isolated the other two members of the Drosophila EXT family genes, which are named sister of tout-velu (sotv) and brother of tout-velu (botv), and encode Drosophila homologues of vertebrate EXT2 and EXT-like 3 (EXTL3), respectively. We show that both Hh and Dpp signalling activities, as well as their morphogen distributions, are defective in cells mutant for ttv, sotv or botv in the wing disc. Surprisingly, although Wg morphogen distribution is abnormal in ttv, sotv and botv, Wg signalling is only defective in botv mutants or ttv-sotv double mutants, and not in ttv nor sotv alone, suggesting that Ttv and Sotv are redundant in Wg signalling. We demonstrate further that Ttv and Sotv form a complex and are co-localized in vivo. Our results, along with previous studies on Ttv, provide evidence that all three Drosophila EXT proteins are required for the biosynthesis of HSPGs, and for the gradient formation of the Wg, Hh and Dpp morphogens. Our results also suggest that HSPGs have two distinct roles in Wg morphogen distribution and signalling.     

Regeneration and transdetermination: The role of wingless and its regulation

Imaginal discs of Drosophila have the remarkable ability to regenerate. After fragmentation wound healing occurs, ectopic wg is induced and a blastema is formed. In some, but not all fragments, the blastema will replace missing structures and a few cells can become more plastic and transdetermine to structures of other discs. A series of systematic cuts through the first leg disc revealed that a cut must transect the dorsal-proximal disc area and that the fragment must also include wg-competent cells. Fragments that fail to both transdetermine and regenerate missing structures will do both when provided with exogenous Wg, demonstrating the necessity of Wg in regenerative processes. In intact leg discs ubiquitously expressed low levels of Wg also leads to blastema formation, regeneration and transdetermination. Two days after exogenous wg induction the endogenous gene is activated, leading to elevated levels of Wg in the dorsal aspect of the leg disc. We identified a wg enhancer that regulates ectopic wg expression. Deletion of this enhancer increases transdetermination, but lowers the amount of ectopic Wg. We speculate that this lessens repression of dpp dorsally, and thus creates a permissive condition under which the balance of ectopic Wg and Dpp is favorable for transdetermination.     

昆虫腹肢发育相关基因的研究进展

昆虫躯干外着生有一系列附属器官,主要包括背侧附器和腹侧附器,其中腹肢的多样性表现尤为突出。腹肢的发育过程受到多种调控因子的作用。本文就腹肢发育相关基因的表达、功能及调控因子间的相互作用等方面进行简要的综述。一方面,腹肢作为整体受Hox基因和成形素基因（Dpp/Wg）的调控,Hox基因直接决定腹肢的有无,Dpp/Wg通过其表达产物形成浓度梯度调控整个腹肢的发育,两者在腹肢整体发育中的作用不可取代。另一方面,腹肢基部、中部及远端部位分别受到各自特异的调控因子的作用。其中hth,tsh及al等均主要调节腹肢基部的发育,dac通过与Dll和Dpp/Wg相互作用从而调节腹肢中部的发育,bab,Dll及Lim1等对腹肢远端发育发挥重要作用。关节的形成对腹肢分节的形成至关重要,Notch信号通路相关因子如配体基因Dl和Ser,修饰物基因fng及下游靶基因odd,sob,drm和bowl等调节该过程。因此,研究昆虫腹肢发育相关基因,对于深入揭示腹肢的发育及其在进化过程中多样性形成的分子机制具有至关重要的作用。     

Gene expression reveals evidence for EGFR‐dependent proximal‐distal limb patterning in a myriapod

Evolution of segmented limbs is one of the key innovations of Arthropoda, allowing development of functionally specific specialized head and trunk appendages, a major factor behind their unmatched evolutionary success. Proximodistal limb patterning is controlled by two regulatory networks in the vinegar fly Drosophila melanogaster, and other insects. The first is represented by the function of the morphogens Wingless (Wg) and Decapentaplegic (Dpp); the second by the EGFR‐signaling cascade. While the role of Wg and Dpp has been studied in a wide range of arthropods representing all main branches, that is, Pancrustacea (= Hexapoda + Crustacea), Myriapoda and Chelicerata, investigation of the potential role of EGFR‐signaling is restricted to insects (Hexapoda). Gene expression analysis of Egfr, its potential ligands, and putative downstream factors in the pill millipede Glomeris marginata (Myriapoda: Diplopoda), reveals that—in at least mandibulate arthropods—EGFR‐signaling is likely a conserved regulatory mechanism in proximodistal limb patterning.     

器官成形素调控果蝇翅芽细胞形貌的研究进展

果蝇翅芽是研究细胞形貌发生的模式系统。在果蝇翅芽的发育过程中,器官成形素由浓度高的区域(成形素表达细胞)向浓度低的区域(接收细胞)移动,形成动态的浓度梯度。器官成形素信号通路的激活调控翅芽细胞的形貌发生、存活、生长和分化。目前已鉴定的在翅芽细胞表达的器官成形素包括Hedgehog(Hh),Decapentaplegic(Dpp)和Wingless(Wg)。结合国际最新研究进展,本文综述了3种器官成形素在翅芽细胞形貌发生过程中的重要作用,讨论了细胞形貌发生的分子机制。     

Retinal morphogenesis in Drosophila: Hints from an eye-specific decapentaplegic allele

decapentaplegic (dpp) regulates many aspects of imaginal disc growth and patterning in Drosophila. We have analyzed the phenotype of an eye-specific dpp allele, dpp^blk, which causes a reduction in the size of the retina due to a loss of ventral ommatidia. Prior to the onset of differentiation, dpp^blk eye discs are normal regarding size, shape, and ability to express dorsal and ventral markers. However, expression of a dpp-lacZ reporter is reduced at the ventral margin. Additional dorsoventral asymmetry appears during retinal differentiation: the morphogenetic furrow (MF) initiates normally at the posterior tip of the disc, but fails to propagate into the ventral epithelium. This defect can be rescued by increasing dpp expression along the ventral margin by local removal of patched function. We propose that the primary defect in dpp^blk is an inability to activate dpp expression properly at the ventral margin. This has two consequences: it prevents initiation from the ventral margin, and it renders the ventral epithelium unresponsive to differentiation signals emanating from the MF. Dev. Genet. 20:197–207, 1997. © 1997 Wiley-Liss, Inc.     

Retinal determination genes as targets and possible effectors of extracellular signals

Retinal determination genes are sufficient to specify eyes in ectopic locations, raising the question of how these master regulatory genes define an eye developmental field. Genetic mosaic studies establish that expression of the retinal determination genes eyeless, teashirt, homothorax, eyes absent, sine oculis, and dachshund are each regulated by combinations of Dpp, Hh, N, Wg, and Ras signals in Drosophila. Dpp and Hh control eyeless, teashirt, sine oculis, and dachshund expression, Dpp and Ras control homothorax, and all the signaling pathways affect eyes absent expression. These results suggest that eye-specific development uses retinal determination gene expression to relay positional information to eye target genes, because the distinct, overlapping patterns of retinal determination gene expression reflect the activities of the extracellular signaling pathways.     

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.