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Developmental patterning is thought to be regulated by conserved signalling pathways. Initial patterns are often broad before refining to only those cells that commit to a particular fate. However, the mechanisms by which pattern refinement takes place remain to be addressed. Using the posterior crossvein (PCV) of the Drosophila pupal wing as a model, into which bone morphogenetic protein (BMP) ligand is extracellularly transported to instruct vein patterning, we investigate how pattern refinement is regulated. We found that BMP signalling induces apical enrichment of Myosin II in developing crossvein cells to regulate apical constriction. Live imaging of cellular behaviour indicates that changes in cell shape are dynamic and transient, only being maintained in those cells that retain vein fate competence after refinement. Disrupting cell shape changes throughout the PCV inhibits pattern refinement. In contrast, disrupting cell shape in only a subset of vein cells can result in a loss of BMP signalling. We propose that mechano-chemical feedback leads to competition for the developmental signal which plays a critical role in pattern refinement.

Maintaining apicobasal polarity (ABP) is crucial for epithelial integrity and homeostasis during tissue development. Although recent studies have greatly advanced our understanding of intracellular mechanisms underlying ABP establishment, it remains largely unknown how the ABP is regulated at the tissue level. Here, we address intercellular mechanisms coordinating ABP using the Drosophila wing imaginal disc. By studying Scribble, a key ABP determinant, we show that ABP is regulated through intercellular alignment, which takes place either progressively or regressively in a context-dependent manner. Cells expressing wild type scribble progressively restore ABP in scribble hypomorphic mutant cells. In contrast, cells with conditional scribble knockdown instigate the regressive loss of polarity in abutting wild type cells. Our data reveal that genetic and physical interactions between Scribble, Septate junction complex and α-Catenin appear to be key for sustaining intercellular network of ABP. Taken together, our findings indicate that the intercellular relay of the status of ABP contributes to the robustness of polarity across the tissue.

Bone morphogenetic proteins (BMPs), members of the transforming growth factor-s (TGF-s) family, have been shown to contribute to embryogenesis and organogenesis during animal development. Relevant studies provide support for the following concepts: (a) BMP signals are evolutionarily highly conserved as a genetic toolkit; (b) spatiotemporal distributions of BMP signals are precisely controlled at the post-translational level; and (c) the BMP signaling network has been co-opted to adapt to diversified animal development. These concepts originated from the historical findings of the Spemann-Mangold organizer and the subsequent studies about how this organizer functions at the molecular level. In this Commentary, we focus on two topics. First, we review how the BMP morphogen gradient is formed to sustain larval wing imaginal disc and early embryo growth and patterning in Drosophila. Second, we discuss how BMP signal is tightly controlled in a context-dependent manner, and how the signal and tissue dynamics are coupled to facilitate complex tissue structure formation. Finally, we argue how these concepts might be developed in the future for further understanding the significance of BMP signaling in animal development.

Developmental patterning is thought to be regulated by conserved signalling pathways. Initial patterns are often broad before refining to only those cells that commit to a particular fate. However, the mechanisms by which pattern refinement takes place remain to be addressed. Using the posterior crossvein (PCV) of theDrosophilapupal wing as a model, into which bone morphogenetic protein (BMP) ligand is extracellularly transported to instruct vein patterning, we investigate how pattern refinement is regulated. We found that BMP signalling induces apical enrichment of Myosin II in developing crossvein cells to regulate apical constriction. Live imaging of cellular behaviour indicates that changes in cell shape are dynamic and transient, only being maintained in those cells that retain vein fate after refinement. Disrupting cell shape changes throughout the PCV inhibits pattern refinement. In contrast, disrupting cell shape in only a subset of vein cells can result in a loss of BMP signalling. In addition, we observed that expressing the constitutively active form of the BMP type I receptor in clones caused apical constriction autonomously and often induced BMP signalling loss in the PCV region in a non-autonomous manner. We propose that the cell shape changes of future PCV cells allow them to compete more efficiently for the basally localised BMP signal by forming a mechano-chemical feedback loop. This study highlights a new form of competition among the cells: competing for a signal that induces cell fate.

Wing venation among insects serves as an excellent model to address how diversified patterns are produced. Previous studies suggest that evolutionarily conserved Decapentaplegic (Dpp)/Bone Morphogenetic Protein (BMP) signal plays a critical role in wing vein development in the dipteran Drosophila melanogaster and the hymenopteran sawfly Athalia rosae. In sawfly, dpp is ubiquitously expressed in the wing during prepupal stages, but Dpp/BMP signal is localized in the future vein cells. Since localized BMP signaling involves BMP binding protein Crossveinless (Cv), redistribution of BMP ligands appears to be crucial for sawfly wing vein formation. However, how ubiquitously expressed ligands lead to a localized signal remains to be addressed. Here, we found that BMP binding protein short gastrulation (Sog) is highly expressed in the intervein cells. Our data also reveal that BMP type I receptors thickveins (Tkv) and saxophone (Sax) are highly expressed in intervein cells and at lower levels in the vein progenitor cells. RNAi knockdown of Ar-tkv or Ar-sax indicates that both receptors are required for localized BMP signaling in the wing vein progenitor cells. Taken together, our data suggest that spatial transcription of core- and co-factors of the BMP pathway sustain localized BMP signaling during sawfly wing vein development. (C) 2018 Elsevier Ltd. All rights reserved.

Significance Tissue morphogenesis is a dynamic process often accompanied by cell patterning and differentiation. Although how conserved growth factor signaling affects cell and tissue shapes has been actively studied, much less is known about how signaling and dynamic morphogenesis are mutually coordinated. Our study shows that BMP signaling and 3D morphogenesis of the Drosophila pupal wing are tightly coupled. These findings are highlighted by the fact that the directionality of BMP signal is changed from lateral planar during the inflation stage to interplanar after re-apposition of the dorsal and ventral wing epithelia. We suspect that the dynamic interplay between planar and interplanar signaling linked to tissue shape changes is likely to be used across species in many developing organs., At the level of organ formation, tissue morphogenesis drives developmental processes in animals, often involving the rearrangement of two-dimensional (2D) structures into more complex three-dimensional (3D) tissues. These processes can be directed by growth factor signaling pathways. However, little is known about how such morphological changes affect the spatiotemporal distribution of growth factor signaling. Here, using the Drosophila pupal wing, we address how decapentaplegic (Dpp)/bone morphogenetic protein (BMP) signaling and 3D wing morphogenesis are coordinated. Dpp, expressed in the longitudinal veins (LVs) of the pupal wing, initially diffuses laterally within both dorsal and ventral wing epithelia during the inflation stage to regulate cell proliferation. Dpp localization is then refined to the LVs within each epithelial plane, but with active interplanar signaling for vein patterning/differentiation, as the two epithelia appose. Our data further suggest that the 3D architecture of the wing epithelia and the spatial distribution of BMP signaling are tightly coupled, revealing that 3D morphogenesis is an emergent property of the interactions between extracellular signaling and tissue shape changes.

Secreted ligands in the Dpp/BMP family drive dorsal-ventral (D/V) axis formation in all Bilaterian species. However, maternal factors regulating Dpp/BMP transcription in this process are largely unknown. We identified the BTB domain protein longitudinals lacking-like (lolal) as a modifier of decapentaplegic (dpp) mutations. We show that Lolal is evolutionarily related to the Trithorax group of chromatin regulators and that lolal interacts genetically with the epigenetic factor Trithorax-like during Dpp D/V signaling. Maternally driven Lolal(HA) is found in oocytes and translocates to zygotic nuclei prior to the point at which dpp transcription begins. lolal maternal and zygotic mutant embryos display significant reductions in dpp, pMad, and zerknullt expression, but they are never absent. The data suggest that lolal is required to maintain dpp transcription during D/V patterning. Phylogenetic data revealed that lolal is an evolutionarily new gene present only in insects and crustaceans. We conclude that Lolal is the first maternal protein identified with a role in dpp D/V transcriptional maintenance, that Lolal and the epigenetic protein Trithorax-like are essential for Dpp D/V signaling and that the architecture of the Dpp D/V pathway evolved in the arthropod lineage after the separation from vertebrates via the incorporation of new genes such as lolal.

SummaryAt the level of organ formation, tissue morphogenesis drives developmental processes in animals, often involving the rearrangement of two-dimensional (2D) structures into more complex three-dimensional (3D) tissues. These processes can be directed by growth factor signaling pathways. However, little is known about how such morphological changes affect the spatiotemporal distribution of growth factor signaling. Here, using theDrosophilapupal wing, we address how Decapentaplegic (Dpp) / Bone Morphogenetic Protein (BMP) signaling and 3D wing morphogenesis are coupled. Dpp, expressed in the longitudinal veins (LVs) of the pupal wing, initially diffuses laterally during the inflation stage to regulate cell proliferation. Dpp localization is then refined to the LVs within each epithelial plane, but with active interplanar signaling for vein patterning, as the two epithelia appose. Our data further suggest that the 3D architecture of the wing epithelia directs the spatial distribution of BMP signaling, revealing how 3D morphogenesis is an emergent property of the interactions between extracellular signaling and tissue shape changes.

Dorsoventral patterning of the Drosophila embryo is regulated by graded distribution of bone morphogenetic proteins (BMPs) composed of two ligands, decapentaplegic (Dpp) a BMP2/4 ortholog and screw (Scw) a BMP5/6/7/8 family member. scwE1 encodes an unusual allele that was isolated as a dominant enhancer of partial loss-of-function mutations in dpp. However, the molecular mechanisms that underlie this genetic interaction remain to be addressed. Here we show that scwE1 contains a mutation at the furin cleavage site within the prodomain that is crucial for ligand production. Furthermore, our data show that ScwE1 preferentially forms heterodimers with Dpp rather than homotypic dimers, providing a possible explanation for the dominant negative phenotype of scwE1 alleles. The unprocessed prodomain of ScwE1 remains in a complex with the Dpp:Scw heterodimer, and thus could interfere with interaction of the ligand with the extracellular matrix, or the kinetics of processing/secretion of the ligand in vivo. These data reveal novel mechanisms by which post-translational regulation of Scw can modulate Dpp signaling activity.

Members of the transforming growth factor-β (TGF-β) family of secreted proteins are present in all multicellular animals. TGF-β proteins are versatile intercellular signalling molecules that orchestrate cell fate decisions during development and maintain homeostasis in adults. The Smad family of signal transducers implements TGF-β signals in responsive cells. Given the ability of TGF-β ligands to induce dramatic responses in target cells, numerous regulatory mechanisms exist to prevent unintended consequences. Here we review new reports of extracellular and post-translational regulation in Drosophila and vertebrates. Extracellular topics include the regulation of TGF-β signalling range and the coordination between tissue morphogenesis and TGF-β signalling. Post-translational topics include the regulation of TGF-β signal transduction by Gsk3-β phosphorylation of Smads and by cycles of Smad mono- and deubiquitylation. Extension of the ubiquitylation data to the Hippo pathway is also discussed.

The pattern of wing venation varies considerably among different groups of insects and has been used as a means of species-specific identification. However, little is known about how wing venation is established and diversified among insects. The decapentaplegic (Dpp)/bone morphogenetic protein (BMP) signaling pathway plays a critical role in wing vein formation during the pupal stages in Drosophila melanogaster. A key mechanism is BMP transport from the longitudinal veins (LVs) to the posterior crossvein (PCV) by the BMP-binding proteins, short gastrulation (Sog) and twisted gastrulation2/crossveinless (Tsg2/Cv). To investigate whether the BMP transport mechanism is utilized to specify insect wing vein patterns in other than Drosophila, we used the sawfly Athalia rosae as a model, which has distinct venation patterns in the fore- and hindwings. Here, we show that Ar-dpp is ubiquitously expressed in both the fore- and hindwings, but is required for localized BMP signaling that reflects distinct wing vein patterns between the fore- and hindwings. By isolating Ar-tsg/cv in the sawfly, we found that Ar-Tsg/Cv is also required for BMP signaling in wing vein formation and retains the ability to transport Dpp. These data suggest that the BMP transport system is widely used to redistribute Dpp to specify wing venation and may be a basal mechanism underlying diversified wing vein patterns among insects.

Epithelial cells are characterized by apical-basal polarity. Intrinsic factors underlying apical-basal polarity are crucial for tissue homeostasis and have often been identified to be tumor suppressors. Patterning and differentiation of epithelia are key processes of epithelial morphogenesis and are frequently regulated by highly conserved extrinsic factors. However, due to the complexity of morphogenesis, the mechanisms of precise interpretation of signal transduction as well as spatiotemporal control of extrinsic cues during dynamic morphogenesis remain poorly understood. Wing posterior crossvein (PCV) formation in Drosophila serves as a unique model to address how epithelial morphogenesis is regulated by secreted growth factors. Decapentaplegic (Dpp), a conserved bone morphogenetic protein (BMP)-type ligand, is directionally trafficked from longitudinal veins (LVs) into the PCV region for patterning and differentiation. Our data reveal that the basolateral determinant Scribbled (Scrib) is required for PCV formation through optimizing BMP signaling. Scrib regulates BMP-type I receptor Thickveins (Tkv) localization at the basolateral region of PCV cells and subsequently facilitates Tkv internalization to Rab5 endosomes, where Tkv is active. BMP signaling also up-regulates scrib transcription in the pupal wing to form a positive feedback loop. Our data reveal a unique mechanism in which intrinsic polarity genes and extrinsic cues are coupled to promote robust morphogenesis., Author Summary Epithelial morphogenesis is one of the key processes in animal development. Evolutionarily conserved growth factors frequently instruct patterning and differentiation in morphogenesis. However, little is known about how extracellular cues and epithelial morphogenesis are mutually coordinated in vivo. Wing posterior crossvein (PCV) development in Drosophila provides an excellent system for understanding how bone morphogenetic protein (BMP) signaling regulates patterning and differentiation of epithelia. We find that the apical-basal polarity gene Scribbled (Scrib) is required for PCV formation by optimizing BMP signaling in the PCV region as follows. First, Scrib regulates BMP type-I receptor Thickveins (Tkv) localization basally. Second, Scrib facilitates Tkv internalization to the Rab5 endosomes to optimize signal transduction after receptor-ligand binding. Third, BMP signaling up-regulates scrib transcription in the pupal wing to form a positive feedback loop. These results suggest that coupling between epithelial polarity genes and conserved growth factors play crucial roles in patterning and differentiation of epithelia.

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(Delta N)), which lacks a short domain at the N-terminus essential for its interaction with Dally. Dpp(Delta N) 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.

In the early Drosophila embryo, Bone morphogenetic protein (BMP) activity is positively and negatively regulated by the BMP-binding proteins Short gastrulation (Sog) and Twisted gastrulation (Tsg). We show here that a similar mechanism operates during crossvein formation, utilizing Sog and a new member of the tsg gene family, encoded by the crossveinless (cv) locus. The initial specification of crossvein fate in the Drosophila wing requires signaling mediated by Dpp and Gbb, two members of the BMP family. cv is required for the promotion of BMP signaling in the crossveins. Large sog clones disrupt posterior crossvein formation, suggesting that Sog and Cv act together in this context. We demonstrate that sog and cv can have both positive and negative effects on BMP signaling in the wing. Moreover, Cv is functionally equivalent to Tsg, since Tsg and Cv can substitute for each other's activity. We also confirm that Tsg and Cv have similar biochemical activities: Sog/Cv complex binds a Dpp/Gbb heterodimer with high affinity. Taken together, these studies suggest that Sog and Cv promote BMP signaling by transporting a BMP heterodimer from the longitudinal veins into the crossvein regions.

SummaryPatterning 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.

Insect wings are great resources for studying morphological diversities in nature as well as in fossil records. Among them, variation in wing venation is one of the most characteristic features of insect species. Venation is therefore, undeniably a key factor of species-specific functional traits of the wings; however, the mechanism underlying wing vein formation among insects largely remains unexplored. Our knowledge of the genetic basis of wing development is solely restricted toDrosophila melanogaster. A critical step in wing vein development inDrosophilais the activation of the decapentaplegic (Dpp)/bone morphogenetic protein (BMP) signalling pathway during pupal stages. A key mechanism is the directional transport of Dpp from the longitudinal veins into the posterior crossvein by BMP-binding proteins, resulting in redistribution of Dpp that reflects wing vein patterns. Recent works on the sawflyAthalia rosae, of the order Hymenoptera, also suggested that the Dpp transport system is required to specify fore- and hindwing vein patterns. Given that Dpp redistribution via transport is likely to be a key mechanism for establishing wing vein patterns, this raises the interesting possibility that distinct wing vein patterns are generated, based on where Dpp is transported. Experimental evidence inDrosophilasuggests that the direction of Dpp transport is regulated by prepatterned positional information. These observations lead to the postulation that Dpp generates diversified insect wing vein patterns through species-specific positional information of its directional transport. Extension of these observations in some winged insects will provide further insights into the mechanisms underlying diversified wing venation among insects.

Bone morphogenetic protein (BMP) signalling regulates embryonic dorsal-ventral cell fate decisions in flies, frogs and fish. BMP activity is controlled by several secreted factors including the antagonists chordin and short gastrulation (SOG). Here we show that a second secreted protein, Twisted gastrulation (Tsg), enhances the antagonistic activity of Sog/chordin. In Drosophila, visualization of BMP signalling using anti-phospho-Smad staining shows that the tsg and sog loss-of-function phenotypes are very similar. In S2 cells and imaginal discs, TSG and SOG together make a more effective inhibitor of BMP signalling than either of them alone. Blocking Tsg function in zebrafish with morpholino oligonucleotides causes ventralization similar to that produced by chordin mutants. Co-injection of sub-inhibitory levels of morpholines directed against both Tsg and chordin synergistically enhances the penetrance of the ventralized phenotype. We show that Tsgs from different species are functionally equivalent, and conclude that Tsg is a conserved protein that functions with SOG/chordin to antagonize BMP signalling.

Diffusible morphogen models have been used widely to explain regional specification of tissues and body axes during animal development. The three-signal model for patterning the dorsal–ventral axis of the amphibian embryo proposes, in part, that a factor(s) secreted from Spemann's organizer is responsible for converting lateral marginal zone into more dorsal cell fates. We examine the possibility that chordin, a secreted inhibitor of bone morphogenetic protein (BMP) signaling and candidate “dorsalizing signal,” is a long-range-acting factor. We show that chordin can, when overexpressed, act directly over distances of at least 450 μm in the early Xenopus embryo to create a gradient of BMP signaling. However, since lower levels of chordin can still induce secondary axes and these amounts of chordin act only locally to inhibit a BMP target gene, we suggest that chordin likely acts as a short-range signal in vivo. Furthermore, BMP1, a secreted metalloprotease that cleaves chordin protein in vitro, inhibits chordin's axis-inducing effects, suggesting that BMP1 functions to negatively regulate chordin's action in vivo. A dominant-negative mutant BMP1 blocks the in vitro cleavage of chordin protein by wild-type BMP1 and induces secondary axes when injected ventrally. We argue that BMP1 and Xolloid are probably functionally redundant metalloproteases and may have two roles in the early Xenopus embryo. One role may be to inhibit the action of low-level chordin protein expressed throughout the entire embryo and a possible second role may be to inhibit activation of a juxtacrine cell relay, thereby confining chordin's action to the organizer region preventing chordin from functioning as a long-range-acting factor.

A variety of extracellular factors regulate morphogenesis during development. However, coordination between extracellular signaling and dynamic morphogenesis is largely unexplored. We address the fundamental question by studying posterior crossvein (PCV) development in Drosophila as a model, in which long-range BMP transport from the longitudinal veins plays a critical role during the pupal stages. Here, we show that RhoGAP Crossveinless-C (Cv-C) is induced at the PCV primordial cells by BMP signaling and mediates PCV morphogenesis cell-autonomously by inactivating members of the Rho-type small GTPases. Intriguingly, we find that Cv-C is also required non-cell-autonomously for BMP transport into the PCV region, while a long-range BMP transport is guided toward ectopic wing vein regions by loss of the Rho-type small GTPases. We present evidence that low level of ß-integrin accumulation at the basal side of PCV epithelial cells regulated by Cv-C provides an optimal extracellular environment for guiding BMP transport. These data suggest that BMP transport and PCV morphogenesis are tightly coupled. Our study reveals a feed-forward mechanism that coordinates the spatial distribution of extracellular instructive cues and morphogenesis. The coupling mechanism may be widely utilized to achieve precise morphogenesis during development and homeostasis., Author Summary It has been extensively studied how tissue morphogenesis is regulated by a variety of extracellular cues. Given that dynamic morphogenesis coincides with arrival of extracellular factors, there must be also mechanisms that coordinate extracellular signaling and intracellular morphogenesis. However, the coordination is largely unknown, due to the complexity of morphogenesis in vivo. We addressed the fundamental question by studying posterior crossvein (PCV) development in Drosophila as a model, in which a long-range transport of bone morphogenetic protein (BMP) type ligands from adjacent longitudinal veins plays a critical role during the pupal stages. Here, we first showed that RhoGAP Crossveinless-C (Cv-C) is induced at the PCV region by BMP signal and mediates PCV morphogenesis. By modulating wing vein morphogenesis, we then found that PCV morphogenesis is required for BMP transport, while ectopic wing vein morphogenesis sufficiently guides a long-range BMP transport. These data suggest a feed-forward mechanism that coordinates the spatial distribution of extracellular instructive cues and morphogenesis. The coupling mechanism may be widely utilized to achieve precise tissue morphogenesis and tissue homeostasis.

When chitin synthase 2 of Saccharomyces cerevisiae was overexpressed in yeast cells using GAL1 promoter, deletion of the N-terminal 193 amino acids significantly increased the level of the protein without affecting its characteristics. We partially purified N-terminally truncated chitin synthase 2 by product entrapment and ion exchange column chromatography, and found that it was active even without trypsin treatment when appropriate divalent cations were present in the reaction mixture. This chitin synthase activity was independent of the N-terminal 193 amino acid truncation, because partially purified full length enzyme also exhibited the activity without trypsin treatment in the presence of appropriate cations. Furthermore, the molecular weights of these two forms of chitin synthase 2 were coincident with those estimated from the deduced amino acid sequence, and most of the chitin synthase 2 in the yeast membrane was present as an unprocessed form, as judged from its molecular weight. Treatment of either full length or truncated enzyme with trypsin, however, further increased the enzyme activity by four to fivefold, and produced a 35 kDa polypeptide that specifically reacted with monoclonal antibody raised against the region containing the putative active site of chitin synthase 2. Thus, it appears that predominant native (unprocessed) chitin synthase 2 is active, but the 35 kDa region encompassing the active site is sufficient for the catalytic activity.

The ability of secreted Transforming Growth Factor β (TGFβ) proteins to act as morphogens dictates that their influence be strictly regulated. Here, we report that maternally contributed fat facets (faf; a homolog of USP9X/FAM) is essential for proper interpretation of the zygotic Decapentaplegic (Dpp) morphogen gradient that patterns the embryonic dorsal-ventral axis. The data suggest that the loss of faf reduces the activity of Medea (a homolog of Smad4) below the minimum necessary for adequate Dpp signaling and that this is likely due to excessive ubiquitylation on a specific lysine. This study supports the hypothesis that the control of cellular responsiveness to TGFβ signals at the level of Smad4 ubiquitylation is a conserved mechanism required for proper implementation of a morphogen gradient.

The bone morphogenetic protein (BMP) family ligand decapentaplegic (Dpp) plays critical roles in wing vein development during pupal stages in Drosophila. However, how the diffusible Dpp specifies elaborate wing vein patterns remains unknown. Here, we visualized Dpp distribution in the pupal wing and found that it tightly reflects the wing vein patterns. We show that Dpp is directionally transported from the longitudinal veins (LVs) into the posterior crossvein (PCV) primordial region by the extracellular BMP-binding proteins, short gastrulation (Sog) and crossveinless (Cv). Another BMP-type ligand, glass bottom boat (Gbb), also moves into the PCV region and is required for Dpp distribution, presumably as a Dpp–Gbb heterodimer. In contrast, we found that most of the Dpp is actively retained in the LVs by the BMP type I receptor thickveins (Tkv) and a positive feedback mechanism. We provide evidence that the directionality of Dpp transport is manifested by sog transcription that prepatterns the PCV position in a Dpp signal-independent manner. Taken together, our data suggest that spatial distribution of Dpp is tightly regulated at the extracellular level by combination of long-range facilitated transport toward the PCV and short-range signaling by active retention in the LVs, thereby allowing diffusible ligands to form elaborate wing vein patterns.

Maturation of bone morphogenetic proteins (BMPs) requires cleavage of their precursor proteins by furin-type proprotein convertases. Here, we find that cleavage sites of the BMP2/4/decapentaplegic (DPP) subfamily have been evolutionary diversified and can be categorized into 4 different types. Cnidaria BMP2/4/DPP is considered to be a prototype containing only 1 furin site. Bilateria BMP2/4/DPP acquired an additional cleavage site with either the combination of minimal–optimal or optimal–optimal furin sites. DPPs belonging to Diptera, such as Drosophila and mosquito, and Lepidoptera of silkworm contain a third cleavage site between the 2 optimal furin sites. We studied how the 3 furin sites (FSI–III) of Drosophila DPP coordinate maturation of ligands and contribute to signals in vivo. Combining mutational analysis of furin-recognition sites and RNAi experiments, we found that the Drosophila DPP precursor is initially cleaved at an upstream furin-recognition site (FSII), with consequent cleavages at 2 furin sites (FSI and FSIII). Both Dfurin1 and Dfurin2 are involved in the processing of DPP proproteins. Biochemical and genetic analyses using cleavage mutants of DPP suggest the first cleavage at FSII to be critical and sufficient for long-range DPP signaling. Our data suggest that the Drosophila DPP precursor is cleaved in a different manner from vertebrate BMP4 even though they are functional orthologs. This indicates that the furin-cleavage sites in BMP2/4/DPP precursors are tolerant to mutations acquired through evolution and have adapted to different systems in diversified species.

Bone morphogenetic proteins (BMPs) are potent inducers of osteoblast differentiation. The accessibility of BMP ligands for binding to their receptors is regulated by secreted proteins Twisted gastrulation (Tsg) and Chordin (Chd). Tsg antagonizes BMP signaling by forming ternary complexes with Chd and BMPs, thereby preventing BMPs from binding to their receptors. In addition to the anti-BMP function, Tsg also has pro-BMP activity, partly mediated by cleavage and degradation of Chd, which releases BMPs from ternary complexes. The roles of Tsg and Chd in osteoblast differentiation are not known. Therefore, in the present study, we investigated the effect of exogenous Tsg and Chd on osteoblast differentiation and mineralization using a well-characterized subclone of MC3T3-E1 osteoblast-like cells. Our results show that Tsg and Chd are expressed in MC3T3-E1 osteoblast-like cells. While Tsg mRNA levels decrease during osteoblast differentiation, Chd levels are found to increase. Tsg and Chd proteins accumulate in the cell culture media as the osteoblasts differentiate. Exogenous Tsg and Chd inhibit osteoblast differentiation and mineralization. Osteocalcin (OCN) mRNA levels decrease following both Tsg and Chd treatment. Tsg and Chd also inhibit alkaline phosphatase (ALP) activity in a dose-dependent manner. To provide insight into the mechanism of Tsg and Chd action, we investigated the effect of Tsg and Chd on BMP activity by determining phosphorylated Smad1 (pSmad1) levels. We show that both Tsg and Chd can independently and in combination reduce pSmad1 levels in MC3T3-E1 cells treated with BMP4. Further, BMP2 partially reverses the inhibitory effect of Tsg and Chd on ALP activity. Taken together, these results suggest that Tsg and Chd are involved in osteoblast differentiation and mineralization by regulating BMP signaling.

Dorsal cell fate in Drosophila embryos is specified by an activity gradient of Decapentaplegic (Dpp), a homologue of bone morphogenetic proteins(Bmps) 2/4. Previous genetic and biochemical studies have revealed that the Sog, Tsg and Tld proteins modify Dpp activity at the post-transcriptional level. The predominant view is that Sog and Tsg form a strong ternary complex with Dpp that prevents it from binding to its cognate receptors in lateral regions of the embryo, while in the dorsalmost cells Tld is proposed to process Sog and thereby liberate Dpp for signaling. In this model, it is not readily apparent how Tld activity is restricted to the dorsal-most cells as it is expressed throughout the entire dorsal domain. In this study, additional genetic and biochemical assays were developed to further probe the relationships between the Sog, Tsg, Tld and Dpp proteins. Using cell based assays, we find that the dynamic range over which Dpp functions for signaling is the same range in which Dpp stimulates the cleavage of Sog by Tld. In addition, our data supports a role for Tsg in sensitizing the patterning mechanism to low levels of Dpp. We propose that the strong Dpp concentration dependence exhibited by the processing reaction, together with movement of Dpp by Sog and Tsg protein can help explain how Tld activity is confined to the dorsal-most region of the embryo through formation of a spatially dependent positive and negative reinforcement loop. Such a mechanism also explains how a sharp rather than smooth signaling boundary is formed.

The human mRNA 5'-capping enzyme cDNA was identified. Three highly related cDNAs, HCE1 (human mRNAcappingenzyme1), HCE1A and HCE1B , were isolated from a HeLa cDNA library. The HCE1 cDNA has the longest ORF, which can encode a 69 kDa protein. A short region of 69 bp in the 3'-half of the HCE1 ORF was missing in HCE1A and HCE1B , and, additionally, HCE1B has an early translation termination signal, which suggests that the latter two cDNAs represent alternatively spliced product. When expressed in Escherichia coli as a fusion protein with glutathione S -transferase, Hce1p displayed both mRNA 5'-triphosphatase (TPase) and mRNA 5'-guanylyltransferase (GTase) activities, and it formed a cap structure at the 5'-triphosphate end of RNA, demonstrating that it indeed specifies an active mRNA 5'-capping enzyme. The recombinant proteins derived from HCE1A and HCE1B possessed only TPase activity. When expressed from ADH1 promoter, HCE1 but not HCE1A and HCE1B complemented Saccharomyces cerevisiae CEG1 and CET1 , the genes for GTase and TPase, respectively. These results demonstrate that the N-terminal part of Hce1p is responsible for TPase activity and the C-terminal part is essential for GTase activity. In addition, the human TPase domain cannot functionally substitute for the yeast enzyme in vivo.

The mRNA-capping enzyme (mRNA 5'-guanylyltransferase) gene was cloned from a Candida albicans genomic DNA library by functional complementation of a Saccharomyces cerevisiae ceg1 delta null mutation. This gene, referred to as CGT1 (C. albicans guanylyltransferase 1), can encode a 52 kDa protein that is highly homologous to S. cerevisiae Ceg1p. CGT1 in a single-copy plasmid complemented the lethality of the S. cerevisiae ceg1 delta null mutation and, like S. cerevisiae Ceg1p, bacterially expressed Cgt1p was able to form a stable complex with the GMP moiety of GTP and to synthesize the cap structure in vitro, demonstrating that CGT1 is the C. albicans mRNA 5'-guanylyltransferase gene. CGT1 seemed to exist as a single copy in the C. albicans genome and was actively transcribed into mRNA. Another ORF was found in an opposite strand very close to the CGT1 locus. This gene shared significant sequence homology with S. cerevisiae FRE1, the gene encoding ferric reductase, and therefore was designated CFL1 (C. albicans ferric-reductase-like gene 1). Despite its sequence homology with S. cerevisiae FRE1, CFL1 mRNA was not induced by iron deprivation, and CFL1 did not complement the slow growth of a S. cerevisiae fre1 delta null mutant in the absence of iron, suggesting that CFL1 is functionally distinct from S. cerevisiae FRE1.