Tsg expression is not altered in dpp mutants (Mason, 1994).
The CBP histone acetyltransferase plays important roles in development and disease by acting as a transcriptional coregulator. A small reduction in the amount of Drosophila CBP (dCBP) leads to a specific loss of signaling by the TGF-ß molecules Dpp and Screw in the early embryo. The expression of Screw itself, and that of two regulators of Dpp/Screw activity, Twisted-gastrulation and the Tolloid protease, is compromised in dCBP mutant embryos. This prevents Dpp/Screw from initiating a signal transduction event in the receiving cell. Smad proteins, the intracellular transducers of the signal, fail to become activated by phosphorylation in dCBP mutants, leading to diminished Dpp/Screw-target gene expression. At a slightly later stage of development, Dpp/Screw-signaling recovers in dCBP mutants, but without a restoration of Dpp/Screw-target gene expression. In this situation, dCBP acts downstream of Smad protein phosphorylation, presumably via direct interactions with the Drosophila Smad protein Mad. It appears that a major function of dCBP in the embryo is to regulate upstream components of the Dpp/Screw pathway by Smad-independent mechanisms, as well as acting as a Smad coactivator on downstream target genes. These results highlight the exceptional sensitivity of components in the TGF-ß signaling pathway to a decline in CBP concentration (Lilja, 2003).
These results suggest that several transcription factors that regulate expression of Dpp/Screw signaling components require the dCBP coactivator for their function in Drosophila embryos, and implicate dCBP in regulation of the Dpp/Screw pathway independently of an interaction with Smad proteins. An additional role of dCBP is to regulate Dpp-target genes, acting at a step downstream of Smad protein phosphorylation. It is likely that direct interactions between dCBP and Mad/Medea contribute to regulation of Dpp target genes). Such interactions have been observed in vitro, both in mammalian systems and using Drosophila proteins. However, a major cause of impaired Dpp/Screw signaling in dCBP mutant embryos is due to reduced tolloid expression. This prevents Dpp/Screw from initiating a signaling event in cells that would normally receive the Dpp/Screw signal, presumably by a failure to cleave the Dpp-Sog and/or Screw-Sog complexes. In fact, a majority of embryos that do not express the Dpp/Screw-target gene rhomboid in dorsal cells, also do not contain phosphorylated Smad proteins. Furthermore, the pattern of phosphorylated Smad proteins correlates closely with that of tolloid expression. For example, in many early, cellularizing dCBP mutant embryos, an anterior patch of both tolloid expression and phosphorylated Smad staining remains. At later stages, tolloid expression recovers in dCBP mutant embryos, as does Dpp/Screw signaling as revealed by Smad protein phosphorylation. This recovery of tolloid expression at later stages of development might explain the recovery of phosphorylated Smad proteins in dCBP mutant embryos, by allowing Dpp/Screw to signal. For these reasons, regulation of tolloid expression appears to be a major means of controlling Dpp/Screw signaling by dCBP (Lilja, 2003).
It is likely that reduced screw expression also contributes to the reduction of phosphorylated Smad proteins observed in dCBP mutant embryos. In both screw and tolloid mutants, phosphorylation of Mad is eliminated. Furthermore, progressive reduction in Screw activity leads to a corresponding progressive deletion of dorsal-most cell fate, the amnioserosa. Tsg is required together with Sog to generate peak Dpp activity in dorsal midline cells. Reduced tsg expression in dCBP mutants may therefore contribute to the lack of Dpp/Screw-target gene expression. However, it is not believe that this lack can explain the defects in dCBP mutants, because in tsg mutants, low levels of Dpp signaling persist in a broad dorsal domain, leading to expanded rhomboid expression in dorsal cells. By contrast, in dCBP mutant embryos, expression of genes in response to a low threshold of Dpp activity, such as U-shaped and the dorsal rhomboid pattern, is eliminated (Lilja, 2003).
These experiments do not address whether dCBP regulation of tolloid, screw, and tsg expression is direct or indirect. However, since expression of these genes begins at about the time when zygotic transcription initiates in the embryo, and the effect of dCBP is evident from the onset of expression of tolloid and screw, the notion is favored that dCBP is acting directly on the enhancers of these genes. It is not yet understood whether HATs such as CBP primarily act to acetylate large chromosomal domains, or are directed to specific genes. In the case of the tolloid gene, the results indicate that dCBP is being recruited to the enhancer by a DNA-binding protein, since the isolated enhancer removed from its normal chromosomal location requires dCBP for its activity (Lilja, 2003).
Given its central position in gene regulation and the great number of mammalian transcription factors shown to interact with CBP, relatively few genes are affected by the dCBP mutation. For example, activation and repression mediated by the Dorsal protein are unaffected in the dCBP mutant embryos, as demonstrated by the expression patterns of Dorsal target genes. Also, no defects in early segmentation gene expression could be observed in germline clone mutants. However, the nej1 mutation used in this study to create dCBP mutant germline clone embryos is a weak mutation that results in a very modest reduction in dCBP levels. Since other means of reducing the dCBP amount by approximately two-fold results in similar gene expression defects, Smad proteins and the unidentified activators of tolloid, tsg, and screw expression are particularly sensitive to a decline in dCBP concentration. It may not be a coincidence that screw, tsg, tolloid, and Dpp-target gene expression are all specifically affected by a small dCBP reduction. Perhaps components of the Dpp/Screw signal transduction pathway have evolved to be coordinately regulated by a common coactivator. Given the phylogenetic conservation of the CBP protein and the TGF-alpha signal transduction pathway, as well as the ability of CBP and Smad proteins to interact in vitro, CBP is likely to play an equally important role in TGF-ß signaling in other metazoans (Lilja, 2003).
Dorsal-ventral patterning in vertebrate and Drosophila embryos requires a conserved system of extracellular proteins to generate a positional information gradient. The components involved include bone morphogenetic proteins (BMP/Dpp), a BMP antagonist (Chordin/Short gastrulation; Chd/Sog) and a secreted metalloproteinase (Xolloid/Tolloid) that cleaves Chd/Sog. Xenopus Twisted gastrulation (xTsg), another member of this signaling pathway, is described. xTsg is expressed ventrally as part of the BMP-4 synexpression group and encodes a secreted BMP-binding protein that is a BMP signaling agonist. The Xenopus homolog of Tsg shares sequence similarities with the cysteine-rich domains of Chordin, and is part of the BMP synexpression group. Biochemical studies show that xTsg and Drosophila dTsg directly bind BMPs with dissociation constants in the low nanomolar range. In microinjection experiments, xTsg mRNA behaves as an agonist of BMP signaling, ventralizing the Xenopus embryo. xTsg competes efficiently with CR1 for binding to BMP and can bind full-length Chordin, forming a ternary complex containing Chordin, BMP and xTsg in vitro. The dorsalizing activity of CR1 is readily competed by wild-type xTsg, and is greatly potentiated by reducing endogenous xTsg activity. The results indicate that xTsg is involved in dorsal-ventral patterning, permitting peak BMP signaling by antagonizing the residual anti-BMP activity of the cleavage products of Chordin. The data suggest a molecular mechanism by which xTsg dislodges latent BMPs bound to Chordin BMP-binding fragments generated by Xolloid cleavage, providing a permissive signal that allows high BMP signaling in the embryo (Oelgeschlager, 2000).
In Drosophila, the phenotype of sog loss-of-function mutants is intriguing: as expected for a Dpp/Scw antagonist, ventral structures are lost but, in addition, the amnioserosa is reduced. This result is paradoxical, as the amnioserosa is the dorsal-most tissue and therefore Sog, a BMP antagonist, is required for maximal BMP signaling. A model proposed to explain the role of Sog in promoting peak Dpp activity suggests that Sog-BMP complexes may permit the diffusion of BMPs originating from more ventral regions, which are then released dorsally by the proteolytic activity of Tolloid. The recent demonstration that BMPs remain bound to individual cysteine-rich domains, which remain intact in the Chordin proteolytic products, makes this interpretation unlikely, unless an additional factor that releases BMP from the cysteine-rich modules is proposed. The twisted gastrulation gene encodes a secreted protein that is specifically required for the differentiation of amnioserosa cells in Drosophila and is a candidate for such a factor (Oelgeschlager, 2000 and references therein).
A full-length xTsg complementary DNA was isolated by using a human expressed sequence tag (EST) to probe a Xenopus gastrula library. The cDNA encodes a protein sharing 41% amino-acid identity with Drosophila Tsg (dTsg), 89% identity with the partial human Tsg sequence and 94% identity with a mouse EST. The xTsg sequence contains a signal peptide, as expected for a secreted protein, and two conserved domains containing multiple cysteines at its amino and carboxy termini. Whole-mount in situ hybridization and polymerase chain reaction with reverse transcription (RT-PCR) show that abundant xTsg maternal transcripts are distributed throughout the animal half of the embryo during cleavage stages. At the late gastrula stage, maternal transcripts decrease and zygotic transcripts appear specifically in the ventral region of the embryo. After neurulation, xTsg transcripts surround ventrally the closed blastopore slit and the neural tube. At the tailbud stage, xTsg transcripts are detected in the postanal region, heart and dorsal eye and closely mimic the expression patterns of BMP-4 and BAMBI, a transmembrane protein that is related to TGF-beta-family type I receptors but lacks an intracellular kinase domain. The postanal region of xTsg expression derives from the ventral-most tissue of the gastrula embryo. It is concluded that xTsg is part of the BMP-4 synexpression group and is expressed in the ventral pole of the embryo (Oelgeschlager, 2000).
Microinjection of xTsg mRNA into each blastomere of the four-cell embryo results in the reduction of dorso-anterior structures. This phenotype is reminiscent of the chordin zebrafish mutant, and of Xenopus embryos microinjected with Xolloid mRNA or with low doses of BMP-4 mRNA. Molecular marker analyses showed that dorsal ectoderm (Sox-2) and dorsal mesoderm (MyoD and Shh) are reduced and that ventral tissues (BMP-4) are expanded in xTsg-injected embryos. It is concluded from these results that xTsg has ventralizing activity (Oelgeschlager, 2000).
To determine whether xTsg functions in the BMP pathway, epistatic analyses were performed by injecting xTsg mRNA into a ventral blastomere together with a dominant-negative BMP receptor (tBR) or extracellular BMP antagonists (noggin, chordin and CR1). xTsg had no effect on the formation of secondary axes by tBR, indicating that it may ventralize the embryo upstream of the BMP receptor. To test whether xTsg could antagonize the dorsalizing activity of the proteolytic fragments of Chordin, a construct was generated containing the N-terminal CR1 domain and terminating at the Xolloid cleavage site. CR1 mRNA induces secondary axes (although at 32-fold higher molar concentrations than those required for full-length chordin mRNA): this induction is blocked by coinjection of xTsg. Xolloid mRNA is able to block the activity of full-length chordin but has no effect on secondary axes induced by the CR1 construct. This indicates that xTsg efficiently antagonizes high doses of CR1 downstream of Xolloid cleavage. The ability of xTsg to inhibit full-length Chordin mRNA presumably results from the activity of uniformly expressed Xolloid proteases in the early embryo. That the ventralizing activity of xTsg requires Xolloid cleavage is confirmed by the observation that a CR1 construct containing 80 additional amino acids downstream of the first Xolloid site could not be antagonized by co-injection of either xTsg or Xolloid mRNAs. The ventralizing activity of xTsg appears to be specific for the Chordin/BMP pathway, since inhibition of BMP by noggin is not affected by co-injection of xTsg mRNA. These epistatic studies are consistent with a model in which xTsg would ventralize the embryo by antagonizing the residual anti-BMP activity of Chordin proteolytic cleavage products (Oelgeschlager, 2000).
When the N-terminal domain of xTsg is compared with the cysteine-rich domains of Chordin, sequence similarities are apparent. Because cysteine-rich domains are BMP-binding modules, a test was performed to see whether epitope-tagged xTsg secreted by transfected 293T cells could bind BMP-4. xTsg does bind to BMP-4 in solution, and this interaction is specific as it can be competed by BMP-2 but not by a 10-fold excess of platelet-derived growth factor, epidermal growth factor, Activin or transforming growth factor (TGF)-beta1. Chemical crosslinking was used to determine whether this molecular interaction is direct. The BMP-binding activity resides in the N-terminal region of xTsg and dTsg, that is, in the domain that shares sequence similarities with the BMP-binding modules of Chordin. It has been concluded that xTsg is a secreted BMP-binding protein that functions in embryos as an agonist of BMP signaling. The other secreted BMP-binding proteins identified, such as Chordin, Noggin, Follistatin and members of the Cerberus family, are antagonists of BMP activity (Oelgeschlager, 2000).
It was next tested whether Chordin and xTsg could compete for BMP binding. Full-length Chordin was immunoprecipitated in the presence of 0.5 nM BMP-4 and increasing amounts of xTsg protein. Instead of competing for the limited amounts of BMP-4, xTsg stimulates binding of BMP-4 to full-length Chordin. Further analyses have showen that xTsg itself can bind to Chordin even in the absence of added BMP. This binding has been confirmed using a cell-culture assay in which permeabilized cells transfected with xTsg cDNA were stained with a Chordin-alkaline phosphatase fusion protein. The binding of xTsg to Chordin requires intact xTsg, since neither the N- nor the C-terminal fragments suffice for the interaction. The observation that the N terminus is unable to bind Chordin but can still bind BMP-4 suggests that these two interactions occur through different sites (Oelgeschlager, 2000).
Crosslinking experiments have showen that xTsg stimulates the binding of BMP-4 to full-length Chordin by forming a trimolecular complex with a relative molecular mass of about 220,000. This shift is consistent with the crosslinking of one dimer of BMP-4 and one dimer of xTsg per Chordin monomer. To investigate the molecular mechanism by which xTsg can antagonize the activity of CR1, equimolar amounts (1 nM) of BMP-4, CR1 and xTsg were incubated for 1 h at room temperature before crosslinking. When CR1 and BMP-4 are incubated together, a complex of Mr 50K is formed, corresponding to the binding of a CR1 monomer to a BMP dimer. Incubation of xTsg and BMP-4 result in the formation of a complex of about 100K, corresponding to a dimer of xTsg bound to a dimer of BMP-4. Remarkably, when CR1, BMP-4 and xTsg are incubated together, only the [xTsg]2[BMP]2 complex is formed. Furthermore, when BMP-4 and CR1 are preincubated for 1 h, BMP-4 can still be dislodged from these preformed complexes by addition of xTsg. It is concluded that, in contrast to full-length Chordin, CR1 bound to BMP-4 is released in the presence of xTsg, resulting in a binary complex of BMP-4 and xTsg (Oelgeschlager, 2000).
To investigate the effect of loss of function of endogenous xTsg, two complementary approaches were used. A construct secreting the C-terminal conserved domain (dn-xTsg) was found to have dominant-negative effects and double-stranded xTsg RNA (RNAi) was also found to interfere with endogenous xTsg function. xTsg RNAi was injected into each animal blastomere at the eight-cell stage together with epitope-tagged xTsg and CR1 mRNAs. RNAi inhibits the expression of secreted xTsg but not of CR1 protein, indicating that RNAi is effective and specific in Xenopus. Microinjection of dn-xTsg or of RNAi results in abnormal development of the postanal region, in particular loss of the ventral fin and shortening of the tail; this is only evident at swimming tadpole stages. At earlier stages of development, only mild defects in the perianal region are observed. However, both dn-xTsg mRNA and RNAi greatly potentiate the dorsalizing effects of low doses of CR1 mRNA injected into all blastomeres of four-cell embryos. These effects could be partially rescued by co-injection of wild-type xTsg mRNA, although the rescue is more effective for dn-xTsg than for xTsg RNAi (Oelgeschlager, 2000).
The Xenopus homolog of Drosophila Tsg is transcribed in the ventral-most region of the embryo. As described, overexpression of this secreted protein ventralizes the embryo through a molecular mechanism functioning upstream of the BMP receptor. Epistasis experiments support a model in which xTsg promotes BMP signaling downstream of Chordin cleavage by the metalloproteinase Xolloid. Chordin is abundantly secreted by the dorsal pole of the embryo, reaching concentrations of 6-12 nM in the extracellular space of Spemann's organizer. Chordin binds BMPs through cysteine-rich modules. Xolloid cleaves Chordin downstream of the two cysteine-rich domains that have the highest affinity for BMP binding. These proteolytic digestion products retain residual BMP-binding activity and can still inhibit the interaction between BMP and its cognate receptor. The results of this study show that xTsg binds BMP directly in the low nanomolar range and will compete effectively with a cysteine-rich module for binding to BMP, leading to the formation of xTsgBMP complexes that permit BMP signaling. In this way, xTsg would promote BMP activity by dislodging the growth factor from an inhibitor in the extracellular space (Oelgeschlager, 2000).
The initial impetus to investigate whether xTsg binds BMP was provided by the observation that the N-terminal conserved domain shares amino-acid similarities with Chordin cysteine-rich repeats. This similarity was found to be functionally significant, since this domain contains the BMP-binding activity of xTsg. Cysteine-rich modules of the Chordin type are found in many extracellular proteins such as von Willebrand factor, thrombospondin, Nel and fibrillar procollagens. The cysteine-rich domain in procollagen IIA binds BMP and TGF-beta1 and is responsible for the dorsalizing activity of procollagen IIA in Xenopus. It is therefore possible that the molecular mechanism described here for xTsg may provide a more general paradigm for signaling in the extracellular space. Similar functions in the release of latent growth factors bound to cysteine-rich domains could be executed by other secreted proteins that share structural similarities with the N-terminal repeat of xTsg, such as members of the connective tissue growth factor (CTGF) and insulin-like growth factor-binding protein (IGFBP) families. In addition, the Cripto and Oep (one-eyed pinhead) proteins, which provide a permissive function for signaling by nodal TGF-beta family members in mouse and zebrafish, share sequence similarities to xTsg in their conserved EGF-like domains (Oelgeschlager, 2000).
In Drosophila, loss of function of Tsg results in the loss of amnioserosa, which requires maximal amounts of Dpp/Scw signaling, but does not affect the rest of the dorsal-ventral pattern. Overexpression of Drosophila Tsg driven by a promoter expressed in the ventral-most region of the embryo rescues dorsal amnioserosa formation in a Drosophila Tsg mutant background, indicating that the protein can diffuse throughout the embryo. Since Drosophila Tsg does not affect other dorsal-ventral fates in Drosophila, it was thought to provide a permissive signal required for amnioserosa differentiation after cells have been exposed to peak levels of Dpp/Scw signaling (Oelgeschlager, 2000 and references therein).
Drosophila Tsg can bind BMPs through its N-terminal domain. Drosophila Sog, although not yet demonstrated to bind Dpp/Scw/BMP in biochemical studies, is cleaved by Tolloid at three sites in the presence of BMP. Two of these cleavage sites are located at similar positions to those in Xenopus Chordin. It is proposed that Drosophila Tsg can act only in the amnioserosa because its function is to release active Dpp/Scw from the cleavage fragments of Sog by Tolloid. Peak signaling would be mediated by Dpp/Scw originating in more ventral regions and reaching the dorsal region by diffusion as a complex with Sog. Release would occur only in the dorsal-most regions in which maximal levels of Tsg, Tolloid, and fragments of Sog bound to BMP are prevalent. In more ventral regions, full-length free Sog would be in excess and would inhibit any released BMPs. This would be particularly true in the presence of Tsg in dorso-lateral regions, in view of the observation that xTsg increases the binding of BMP to full-length Chordin. This model has the attraction of explaining the paradoxical effects of Sog, which inhibits Dpp/Scw signaling in ventral ectoderm but promotes it in the most dorsal regions of the embryo (Oelgeschlager, 2000 and references therein).
By introducing Drosophila Tsg as an additional factor that permits the release of Dpp/Scw from inactive complexes in the amnioserosa, it is not necessary to invoke positive (that is, non-inhibitory) signaling effects of Sog proteolytic cleavage products to account for the Tolloid-dependent long-range effects of Sog diffusion on peak Dpp signaling (Oelgeschlager, 2000 and references therein).
One of the great surprises of developmental genetics has been the evolutionary conservation of molecular mechanisms. The best known example is the discovery that conserved sets of Hox genes determine the anteriorposterior axis in all bilateral animals. The activities and expression patterns of Dpp/BMP-4, Sog/Chordin and Tolloid/Xolloid have led to the view that the dorsalventral axis has been inverted in the course of evolution. Drosophila Tsg, a gene expressed in the dorsal-most blastoderm, has a vertebrate homolog expressed in the ventral-most tissues of the Xenopus embryo. xTsg is part of an intricate extracellular signaling pathway in which the two poles of the dorsal-ventral pattern are defined by sources of secreted Sog/Chordin and dTsg/xTsg on opposite sides of the embryo. The results are consistent with the idea that the ventral side of the arthropod is homologous to the dorsal side of the vertebrate (Oelgeschlager, 2000 and references therein).
Developmental patterning relies on morphogen gradients, which generally involve feedback loops to buffer against perturbations caused by fluctuations in gene dosage and expression. Although many gene components involved in such feedback loops have been identified, how they work together to generate a robust pattern remains unclear. The network of extracellular proteins that patterns the dorsal region of the Drosophila embryo by establishing a graded activation of the bone morphogenic protein (BMP) pathway has been studied. The BMP activation gradient itself is robust to changes in gene dosage. Computational search for networks that support robustness shows that transport of the BMP class ligands (Scw and Dpp) into the dorsal midline by the BMP inhibitor Sog is the key event in this patterning process. The mechanism underlying robustness relies on the ability to store an excess of signaling molecules in a restricted spatial domain where Sog is largely absent. It requires extensive diffusion of the BMP-Sog complexes, coupled with restricted diffusion of the free ligands. Dpp is shown experimentally to be widely diffusible in the presence of Sog but tightly localized in its absence, thus validating a central prediction of a theoretical study (Eldar, 2002).
Graded activation of the BMP pathway subdivides the dorsal region of Drosophila embryos into several distinct domains of gene expression. This graded activation is determined by a well-characterized network of extracellular proteins, which may diffuse in the perivitelline fluid that surrounds the embryo. The patterning network is composed of two BMP class ligands (Scw and Dpp), a BMP inhibitor (Sog), a protease that cleaves Sog (Tld) and an accessory protein (Tsg), all of which are highly conserved in evolution and are used also for patterning the dorso-ventral axis of vertebrate embryos. Previous studies have suggested that patterning of the dorsal region is robust to changes in the concentrations of most of the crucial network components. For example, embryos that contain only one functional allele of scw, sog, tld or tsg are viable and do not show any apparent phenotype. Misexpression of scw or of tsg also renders the corresponding null mutants viable (Eldar, 2002).
To identify the mechanism underlying robustness, a general mathematical model of the dorsal patterning network was formulated. For simplicity, initial analysis was restricted to a single BMP class ligand (Scw or Dpp), a BMP inhibitor (Sog) and the protease (Tld). The general model accounted for the formation of the BMP-Sog complex, allowed for the diffusion of Sog, BMP and BMP-Sog, and allowed for the cleavage of Sog by Tld, both when Sog is free and when Sog is associated with BMP. Each reaction was characterized by a different rate constant (Eldar, 2002).
Extensive simulations were carried out to identify robust networks. At each simulation, a set of parameters (rate constants and protein concentrations) was chosen at random and the steady-state activation profile was calculated by solving three equations numerically. A set of three perturbed networks representing heterozygous situations was then generated by reducing the gene dosages of sog, tld or the BMP class ligand by a factor of two. The steady-state activation profiles defined by those networks were solved numerically and compared with the initial, nonperturbed network. A threshold was defined as a given BMP value (corresponding to the value at a third of the dorsal ectoderm in the nonperturbed network). The extent of network robustness was quantified by measuring the shift in the threshold for all three perturbed networks. Over 66,000 simulations were carried out, with each of the nine parameters allowed to vary over four orders of magnitude (Eldar, 2002).
As expected, in most cases (97.5%) the threshold position in the perturbed networks was shifted by a large extent (>50%). In most of those nonrobust cases, the BMP concentration was roughly uniform throughout the dorsal region. By contrast, Sog was distributed in a concentration gradient with its minimum in the dorsal midline, defining a reciprocal gradient of BMP activation. Thus, the key event in this nonrobust patterning mechanism is the establishment of a concentration gradient of Sog, which was governed by diffusion of Sog from its domain of expression outside the dorsal region, coupled with its cleavage by Tld inside the dorsal region. Although such a gradient has been observed, it is also compatible with other models (Eldar, 2002).
A small class of networks (198 networks, 0.3%) was identified in which a twofold reduction in the amounts of all three genes resulted in a change of less than 10% in the threshold position. Notably, in all of these robust cases, BMP was redistributed in a sharp concentration gradient that peaked in the dorsal midline. In addition, this concentration gradient decreases as a power-low distribution with an exponent n = 2, which indicates the uniqueness of the robust solution. In these cases, Sog was also distributed in a graded manner in the dorsal region. Analysis of the reaction rate constants of the robust networks showed a wide range of possibilities for most parameters. But two restrictions were apparent and defined the robust network design: (1) in the robust networks the cleavage of Sog by Tld was facilitated by the formation of the complex Sog-BMP; (2) the complex BMP-Sog was broadly diffusible, whereas free BMP was restricted (Eldar, 2002).
To identify how robustness is achieved, an idealized network was considered by assuming that free Sog is not cleaved and that free BMP does not diffuse. The steady-state activation profile defined by this network can be solved analytically; the solution reveals two aspects that are crucial for ensuring robustness. First, the BMP-Sog complex has a central role, by coupling the two processes that establish the activation gradient: BMP diffusion and Sog degradation. This coupling leads to a quantitative buffering of perturbations in gene dosage. Second, restricted diffusion of free BMP enables the system to store excess BMP in a confined spatial domain where Sog is largely absent. Changes in the concentration of BMP alter the BMP profile close to the dorsal midline but do not change its distribution in most of the dorsal region (Eldar, 2002).
The complete system, comprising Sog, Tld, Tsg, both Scw and Dpp, and their associated receptors was examined next. Two additional molecular assumptions are required to ensure the robustness of patterning. First, Sog can bind and capture the BMP class ligands even when the latter are associated with their receptors. Second, Dpp can bind Sog only when the latter is bound to Tsg. Indeed, it has been shown that, whereas Sog is sufficient for inhibiting Scw, both Tsg and Sog are required for inhibiting Dpp. This last assumption implies that Tsg functions to decouple the formation of the Scw gradient from the parallel generation of the Dpp gradient, ensuring that Scw and Dpp are transported to the dorsal midline independently by two distinct molecular entities (Eldar, 2002).
The complete model was solved numerically for different choices of rate constants. In particular, the effect of twofold changes in gene dosage was assessed. The steady-state activation profiles can be superimposed, indicating the robustness of the system. In addition, with the exception of Dpp, the expression of all other crucial network components can be altered by at least an order of magnitude before an effect on the position of a given threshold is observed. In the model, the lack of robustness to Dpp stems from its insufficient dosage. Note that the time taken to reach steady state is sensitive to these concentrations of protein. For the wide range of parameters that were used, however, the adjustment time does not exceed the patterning time. Flexible adjustment time thus facilitates the buffering of quantitative perturbations (Eldar, 2002).
This analysis has identified two principle molecular features that are essential for robust network design: first, free Sog is not cleaved efficiently -- an assumption that is supported by the in vitro finding that Sog cleavage by Tld requires BMP; second, the diffusion of free BMP is restricted. This is the central prediction of the theoretical study, namely, that Scw diffusion requires Sog, whereas Dpp diffusion requires both Sog and Tsg. Although several reports suggest that in wild-type embryos both Dpp and Scw are widely diffusible, their ability to diffuse in a sog or tsg mutant background has not been examined as yet (Eldar, 2002).
To monitor the diffusion of Scw or Dpp, the even-skipped (eve) stripe-2 enhancer (st2) was used to misexpress Dpp or Scw in a narrow stripe perpendicular to the normal BMP gradient. In transgenic embryos, dpp or scw RNA was detected in a stripe just posterior to the cephalic furrow. Initially the stripe was about 12 cells wide at early cleavage cycle 14, but refined rapidly to about 6 cells by late cycle 14. The st2-dpp and st2-scw embryos were viable, despite the high expression of these proteins as compared with their endogenous counterparts (Eldar, 2002).
The activation of the BMP pathway was monitored either by staining for pMad or by following dorsal expression of the target gene race, which requires high activation. Scw is a less potent ligand than is Dpp. This experimental setup could not be used to study Scw diffusion properties because expressing st2-scw did not alter the pattern of pMad or race expression in wild-type or sog-/- embryos. By contrast, expression of st2-dpp led to an expansion of both markers in a region that extends far from the st2 expression domain, indicating a wide diffusion of Dpp in a wild-type background. Conversely, on expression of st2-dpp in sog-/- or in tsg-/- embryos, both markers were confined to a narrow stripe in the st2 domain. The width of this stripe was comparable to that of st2-dpp expression, ranging from 6 to 12 cells, indicating that Dpp does not diffuse from its domain of expression in the absence of Sog or Tsg. Taken together, these results show that both Sog and Tsg are required for Dpp diffusion, as predicted by the theoretical analysis (Eldar, 2002).
The computation ability of biochemical networks is striking when one considers that they function in a biological environment where the amounts of the network components fluctuate, the kinetics is stochastic, and sensitive interactions between different computation modules are required. Studies have examined the effect of these properties on cellular computation mechanisms, and robustness has been proposed to be a 'design principle' of biochemical networks. The applicability of this principle to morphogen gradient patterning has been shown during early development. Quantitative analysis can be used to assess rigorously the robustness of different patterning models and to exclude incompatible ones. The remaining, most plausible model points to crucial biological assumptions and serves to postulate the central feedback mechanisms. Applying the same modelling principles to other systems might identify additional 'design principles' that underlie robust patterning by morphogen gradients in development (Eldar, 2002).
BMP signaling is modulated by a number of extracellular proteins, including the inhibitor Chordin, Tolloid-related enzymes (Tld), and the interacting protein Twisted Gastrulation (Tsg). Although in vitro studies have demonstrated Chordin cleavage by Tld enzymes, its significance as a regulatory mechanism in vivo has not been established in vertebrates. In addition, Tsg has been reported in different contexts to either enhance or inhibit BMP signaling through its interactions with Chordin. The zebrafish gastrula has been used to carry out structure/function studies on Chordin, by making versions of Chordin partially or wholly resistant to Tld cleavage and introducing them into chordin-deficient embryos. The cleavage products generated in vivo from wild-type and altered Chordins were examined, and their efficacy as BMP inhibitors was tested in the embryo. Tld cleavage is shown to be crucial in restricting Chordin function in vivo, and is carried out by redundant enzymes in the zebrafish gastrula. Evidence is presented that partially cleaved Chordin is a stronger BMP inhibitor than the full-length protein, suggesting a positive role for Tld in regulating Chordin. Depletion of embryonic Tsg leads to decreased BMP signaling, and to increased levels of Chordin. Finally, it was shown that Tsg also enhances BMP signaling in the absence of Chordin, and its depletion can partially rescue the chordin mutant phenotype, demonstrating that important components of the BMP signaling pathway remain unidentified (Xie, 2005).
Dorsal cell fate in Drosophila embryos is specified by an activity gradient of Decapentaplegic. 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, since 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, it has been found 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, the data support a role for Tsg in sensitizing the patterning mechanism to low levels of Dpp. It is proposed 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 (Shimmi, 2003).
According to the prevailing view, Sog, Tsg and Tld act to create a transport mechanism that helps promote Dpp diffusion from lateral regions of the embryos towards the dorsal side. According to this model, Sog would diffuse into the dorsal domain from its ventral lateral site of synthesis and capture Dpp, thereby preventing Dpp from binding to receptor. Net flux of Sog towards the dorsal side is envisioned to help transport Dpp and thereby increase its concentration in the dorsalmost tissue, which is destined to become the amnioserosa. Tld acts to liberate Dpp by cleaving Sog, and Dpp once released, will either be recaptured by another Sog molecule or bound to its receptors (Shimmi, 2003).
In order for the transport model to produce a Dpp concentration peak, the proper balance between binding affinities, diffusion rates and proteolytic processing is needed. Tsg has been suggested to have several activities that could influence this balance. In one model, Tsg would act to slow down the intrinsic rate of Sog cleavage by Tld. In this case, loss of Tsg is predicted to result in elevated processing of Sog. This should produce a sog loss-of-function phenotype, as is observed when molecular markers are examined. That data argues strongly against this possibility. First, it has been demonstrated that Tsg function is epistatic to Tld. If the tsg mutant phenotype is caused by excess Tld activity, then eliminating Tld should produce a tld loss-of-function phenotype. However, a tsg-like phenotype is observed where there is a general lowering and flattening of the Dpp activity gradient, as assayed by marker gene expression. In addition, biochemical studies reveal that Tsg actually enhances the ability of Tld to cleave Sog. Taken together, it is concluded that Tsg does not function during DV patterning to retard Tld proteolytic activity (Shimmi, 2003).
A second property has been attributed to Tsg: it alters the selection of Tld cleavage sites in Sog thereby producing novel Sog fragments with unique properties. In particular, a Sog fragment termed Supersog containing the first CR domain and a region of the spacer between CR1 and CR2 appears to be produced in vitro by the action of Tsg and Tld. Although the production of Supersog-like fragments are seen under the present reaction conditions described in this study, no enhancement in their production is seen upon Tsg addition. This may reflect loss of an unidentified component during purification or differences in the sensitivities of the CR1 antibodies used in the two studies. These issues are presently under examination. Whether Supersog-type molecules contribute to DV patterning in vivo is unclear. The fact that overexpression of Supersog can partially rescue tsg mutant embryos suggests that they could be important. A full resolution of the role of Supersog will need to await the results of in vivo rescue experiments employing mutants of the different Sog cleavage sites, especially those that lead to the production of Supersog-like fragments (Shimmi, 2003).
One of the primary findings in this report is that the rate of Sog cleavage is very sensitive to the level of the Dpp protein and varies substantially over a 10-fold range. Interestingly, this is the same Dpp concentration range within which low to maximal signaling occurs in S2 cell culture. Tsg sensitizes the system such that both the binding of Dpp to Sog as well as the rate of cleavage of Sog by Tld is stimulated by Tsg protein. Because in the invertebrate system, the binding of ligand to Sog is required for efficient processing of Sog, it is not surprising that the rate of Sog processing goes up in the presence of Tsg. This follows because, at a given concentration of Sog and Dpp, more complex will be formed in the presence of Tsg leading to a higher substrate concentration for the Tld protease. It is speculated that this system evolved in part to enable the embryo to produce a patterning mechanism that functions within the context of a very short developmental window. In Drosophila, the time between initial transcription of dpp during the early blastoderm stage and assignment of fate required for proper gastrulation is only about 40 minutes. In this short time-window, Dpp concentration must reach an effective signaling level. However, using a genomic Dpp-HA construct, it has been possible to visualize Dpp in the early embryo and it is present at much lower levels than in other tissues, such as the epidermis, at later stages of embryogenesis. It is proposed that under these conditions of low Dpp concentration, the presence of Tsg is required to enable Sog to bind to Dpp and to stimulate Sog cleavage in order to create a cyclic binding and release process that enables Dpp to be carried towards the dorsal midline. Furthermore, it is proposed that the intrinsic sensitivity of the cleavage reaction to the Dpp concentration is crucial for formation of a sharp signaling boundary. Thus, as the Dpp concentration drops in the lateral regions as a consequence of Dpp movement towards the dorsal side, the rate of Sog cleavage drops, allowing more Sog to enter this region and further reducing signaling in lateral regions. The movement of Dpp will simultaneously raise Dpp concentration in the dorsal region, further stimulating cleavage and clearance of Sog and thereby reinforcing Dpp signaling at the dorsal midline. This built-in positive and negative reinforcement mechanism should help establish sharp signaling boundaries by formation of steep ligand gradients, instead of the more gradual gradients that would form if Sog cleavage was not sensitive to the Dpp concentration (Shimmi, 2003).
In some vertebrate systems, DV patterning mechanisms have been conserved with respect to the molecules employed, but the polarity of axis over which they act has been inverted. Thus, in both amphibians and zebrafish, Bmp ligands specify ventral cell fates, whereas Bmp inhibitors, such as Chordin, are secreted from dorsal cells. In each of these systems, Tsg- and Tld-like proteins also contribute to axis formation, but the biochemical details of their associations appear different from those found in Drosophila. Two distinctions are most apparent and these probably have biological significance with respect to the patterning mechanism employed by these organisms. In Xenopus, the affinity of chordin for Bmps is significantly higher than Sog for Dpp; Bmps can be coimmunoprecipitated by chordin alone whereas this is not the case for the Drosophila components. In addition, once cleaved by Xolloid, at least some of the CR1 containing fragments of chordin continue to have significant affinity for the Bmp ligand preventing it from signaling (Shimmi, 2003 and references therein).
The second major difference between the Drosophila and Xenopus systems is that the Drosophila processing of Sog is dependant on prior binding of Sog to Dpp, while in Xenopus this is not the case. Rather, Chordin cleavage by Xolloid appears to be constitutive and is not enhanced by any tested ligand. Without ligand dependent cleavage, net movement of Bmps by Chordin diffusion may not readily occur nor would there be a mechanism to both positively and negatively reinforce the processing reaction. Indeed, recent studies have demonstrated that in the Drosophila embryo, Chordin does not have the ability to promote Dpp signaling at a distance, whereas Sog does. As a result, spatially enhanced Bmp concentrations and sharp signaling boundaries that result from net ligand movement by the activities of the Chordin, Xolloid and Tsg proteins may not occur in Xenopus. In fact there is no evidence in Xenopus that loss of Chordin activity actually results in a reduction in Bmp signaling in select regions of the embryo as occurs in Drosophila (Shimmi, 2003).
Despite these differences, Tsg may, nevertheless, play both positive and negative roles in modulating Bmp signaling; however, its mechanism is somewhat different. As processed fragments of Chordin still have reasonable affinity for ligand, they may need to be dislodged to allow for signaling. Tsg binding to Bmps appears to help promote this dislodgment and their ultimate degradation. In Drosophila, since Sog binds poorly to ligand in the absence of Tsg there is no need for Tsg to help promote dissociation of Sog fragments. Rather, it is its ability to help promote association of Sog with Dpp that is key to understanding its function. Tsg appears also to alter the rate of chordin proteolysis. Thus, at a high Tsg-to-chordin ratio, Chordin may be degraded and in this way Tsg might help promote signaling. It is possible that some combination of these properties is used in other vertebrates. For example, in zebrafish it has recently been shown that loss of chordin can enhance a phenotype that results from haplo-insufficiency for swirl, a gene that encodes Bmp2b. This paradoxical observation, that loss of an inhibitor exacerbates a phenotype resulting from loss of a ligand, is exactly analogous to the case of amnioserosa development in Drosophila where loss of Sog (an inhibitor) leads to less Dpp signaling in the dorsal domain. Detailed studies examining the ligand dependence of Chordin cleavage in zebrafish by minifin, the gene encoding a Tld homolog, have not been reported. It is possible therefore, that like Drosophila, this system may also employ a transport mechanism involving Tsg, Chordin and Tld that acts to boost Bmp signaling in specific tissues. It is interesting to note that the mouse homologs of Tsg, Chordin and Tld also exhibit their own distinct biochemical properties. Thus, a new Tld processing site in Chordin is induced by the presence of Tsg but this is not seen when the Xenopus components are used. Thus, it seems probable that the inherent complexity of this multi-component regulatory mechanism has provided numerous targets for evolutionary change. It is speculated that these changes account for the remarkable diversity that this mechanism exhibits with respect to the actual details by which it regulates Bmp signaling in different organisms (Shimmi, 2003).
The Drosophila short gastrulation gene encodes a large extracellular protein (Sog) that inhibits signaling by BMP-related ligands. Sog and its vertebrate counterpart Chordin contain four copies of a cysteine repeat (CR) motif defined by 10 cysteine residues spaced in a fixed pattern and a tryptophan residue situated between the first two cysteines. This study presents a structure-function analysis of the CR repeats in Sog, using a series of deletion and point mutation constructs, as well as constructs in which CR domains have been swapped. This analysis indicates that the CR domains are individually dispensable for Sog function but that they are not interchangeable. These studies reveal three different types of Sog activity: intact Sog, which inhibits signaling mediated by the ligand Glass bottom boat (Gbb), a more broadly active class of BMP antagonist referred to as Supersog, and a newly identified activity, which may promote rather than inhibit BMP signaling. Analysis of the activities of CR swap constructs indicates that the CR domains are required for full activity of the various forms of Sog but that the type of Sog activity is determined primarily by surrounding protein sequences. Cumulatively, this analysis suggests that CR domains interact physically with adjacent protein sequences to create forms of Sog with distinct BMP modulatory activities (Yu, 2004).
The Sog CR domains are defined by a set of 10 cysteine residues with a conserved spacing and a single tryptophan residue located between the first two cysteines. The function of the tryptophan residues was examined by mutating them individually or all to alanine. The finding that all four single W --> A mutants have wild-type Sog function as assayed by misexpression in the wing, either alone or when coexpressed with Tsg, indicates that none of these residues is individually essential for either Sog or Tsg + Sog (Supersog) activities. This finding is also consistent with the results of deleting the individual CR domains. When all four tryptophans were mutated to alanine, however, the Sog-like activity remained relatively unaffected but this mutant was greatly compromised in its ability to interact with Tsg to generate a Supersog-like activity. These results suggest that the tryptophan residues in two or more CRs can mediate functional interaction with Tsg and that Sog residues outside of the four conserved tryptophans are not sufficient on their own to mediate this interaction. However, deletion of the stem region also eliminates the functional Tsg interaction since a mutant lacking the stem and CR1 fails to interact with Tsg while a mutant lacking just CR1 interacts fully. The requirement for the stem region in interacting with Tsg is consistent with both CR and stem sequences being essential for Supersog activity (Yu, 2004).
Truncated forms of Sog consisting of CR1, the stem, and CR2 behave differently from either Sog or Supersog constructs when misexpressed in the wing or embryo. The strongest form of this novel Sog activity is observed when both CR3 and CR4 are deleted (e.g., SogCR1,2). Several lines of evidence suggest that Sog CR1,2 functions by promoting BMP signaling. (1) The effect of misexpressing SogCR1,2 on gene expression in wing discs is most similar to that of misexpressing an activated Sax receptor or the putative Sax ligand Gbb. This profile of gene response is quite distinct from that resulting from misexpression of activated or dominant-negative forms of the BMP receptor or EGF-receptor pathway, which is the other major signaling system regulating early vein development. (2) Misexpression of SogCR1,2 in pupal wings by heat shock results in significant ectopic expression of the rho gene, which is a good measure of BMP vs. EGF-R pathway activation during this stage. (3) Expression of SogCR1,2 in the early embryo broadens the dorsal expression domain of the BMP target gene zen (Yu, 2004).
One attractive model for a positive function of Sog such as that potentially mediated by SogCR1,2 is that in addition to binding to BMPs and preventing them from gaining access to the receptors, Sog might also act as a carrier of BMPs to either protect them from degradation or possibly transport them dorsally. Since the CR3 and CR4 domains appear to be those most critical to inhibition of Gbb/Scw activity, it is possible that the apparent positive activity of SogCR1,2 is the result of removing the Scw/Gbb inhibitory activity, while leaving a carrier function intact. The difference between the activity of SogCR1,2 and Supersog molecules, which lack the CR2 domain, might be explained if CR1 and CR2 can interact in SogCR1,2 to prevent CR1 and/or adjacent sequences from interacting with Dpp, thus neutralizing this remaining potential BMP inhibitory activity. It is currently unclear what relationship, if any, the SogCR1,2 activity has to the positive function of Sog required to sustain dorsal expression of race in the early embryo. SogCR1,2, unlike intact Sog, is unable to rescue race at a distance, but can broaden dorsal zen expression, which intact Sog does not do. The apparent differences between these activities may be the result of threshold-dependent effects in the early embryo or may reflect a fundamentally different mode of action (Yu, 2004).
An important question is whether truncated forms of Sog similar to SogCR1,2 or SogDeltaCR4 are generated and function in vivo. It is known that Tld can cleave Sog in vitro to generate products of approximately the same size as these constructs, and Sog-reactive bands of approximately the same size are observed in early embryos and pupal wings. In addition, forms of Chordin similar in structure to SogCR1,2 and Supersog are produced in humans as the result of alternative RNA splicing . Further analysis of the production and activity of SogCR1,2-like molecules will be required to determine the relevance of such forms in vivo and to determine the mechanism by which they may act on the BMP pathway (Yu, 2004).
Analysis of Sog mutants in which individual CRs are deleted or single conserved tryptophan residues are mutated to alanine suggests that the CR domains perform partially overlapping functions since none of them is absolutely essential for intact Sog activity (e.g., inhibition of Gbb/Scw) or for interaction with Tsg to create a Dpp inhibitory activity. Nonetheless, these experiments also suggest that the CR domains are not equivalent. For example, deletion of CR3 or CR4 had much greater effects in reducing the SOG-like activity than did deletion of CR1 or CR2. Moreover, the fact that Supersog, which contains only CR1, can inhibit Dpp while SogCR4 apparently interferes selectively with Gbb, suggested that CR1 might bind Dpp while CR4 bound Gbb. The results of the CR swap experiments performed in the contexts of SogCR4, Supersog1, and SogCR1,2 are quite informative in resolving this question and provide a surprising answer, namely that sequences adjacent to CR domains are the primary determinants of BMP specificity (Yu, 2004).
While replacing a CR domain in swap constructs typically resulted in greatly reduced activity or inactivity of UAS-transgenes tested as single- or multicopy insertions, those that had activity generated phenotypes similar to those of the parent constructs. These data suggest that the CRs are required in a context-specific fashion to boost the activity levels of the various forms of Sog. These findings, although limited in scope, suggest that the primary determinant of the quality of Sog activity lies within the sequences surrounding the CRs rather than within the CR domains themselves. These non-CR sequences may correspond to identified repetitive motifs such as the SR repeats or the circularly permuted SOG/CHRD domains. The simplest interpretation of this result is that CRs contribute to defining the strength and surrounding sequences determine the specificity of Sog activities. This model of Sog function is consistent with the finding that deletion of any single CR does not eliminate Sog-like activity (e.g., inhibition of Scw/Gbb) or the ability to interact with Tsg (e.g., inhibition of Dpp) and that both the CR1 domain and adjacent stem sequences are required for Supersog activity. The fact that the conserved tryptophans in the CR domains are required in aggregate, but not individually, for interaction with Tsg and that stem sequences are also required for this interaction lends additional support to the view that the CRs function in a partially redundant fashion in conjunction with non-CR sequences. One potential explanation for the results reported here is that the CR domains alone bind to BMPs, but do so with little selectivity. The surrounding sequences may provide specificity by interacting with only a subset of BMPs, thereby increasing the affinity of adjacent CRs for particular BMPs. Alternatively, surrounding sequences may alter the conformation of CR domains or sterically limit their interactions with BMPs, rendering them more selective. Further biochemical analysis will be required to determine the degree to which CRs affect affinity of the various Sog forms for particular BMPs and the mechanism by which surrounding sequences may confer specificity for binding particular BMPs (Yu, 2004).
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. Tsg and Sog play essential roles in transporting Dpp to the dorsal-most cells. Furthermore, biochemical and genetic evidence is presented 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. It is proposed 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, it is demonstrated 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 (Shimmi, 2005).
The suggestion that the facilitated transport of a BMP signaling molecule might be the primary mechanism that generates pattern within the dorsal domain of the Drosophila blastoderm embryo (Holley, 1996) was a conceptual breakthrough, since it could account for the paradoxical abilities of Sog and Tsg to have both positive and negative effects on patterning. However, there was no direct evidence that either Dpp or Scw actually concentrated to the midline. In addition, it did not explain the roles of Dpp and Scw in producing the restricted high-level signaling output at the midline, as measured by p-Mad accumulation, nor did it explain how a lower level of signal was maintained in the more lateral regions to help fate the future dorsal ectoderm. Lastly, it was not apparent how the system achieves resiliency to changes in gene dosages of certain components. The experimental and computational observations described in this study have addressed these issues (Shimmi, 2005).
One of the primary findings is that Dpp and Scw form heterodimers both in tissue culture and in vivo and that these heterodimers are able to synergistically stimulate phosphorylation of Mad in cell culture. Since the Dpp/Scw heterodimers have highest affinity for Sog and Tsg, it is inferred that the heterodimer is the primary ligand transported dorsally by Sog and Tsg, resulting in high levels of p-Mad accumulation at the dorsal midline just prior to gastrulation. Consistent with this view, it was found that Dpp localization to the midline depends on Scw (Shimmi, 2005).
In addition to heterodimers being the preferred translocated species, the heterodimer model also explains the mechanism by which Scw contributes to dorsal patterning. This issue has been enigmatic since scw and its receptor, Sax, are expressed ubiquitously in the early embryo, yet signal output is limited to dorsal cells. In addition, misexpression of Scw or activated Sax produces very limited effects in most tissues, while misexpression of Dpp or activated Tkv results in very dramatic consequences. A partial resolution to this issue was suggested by the finding that coexpression of activated Sax and activated Tkv in embryos or imaginal discs produces a synergistic signal, implying that both the Sax and Tkv signals are necessary for a robust output. However, it has remained unclear whether endogenous, nonactivated receptors can produce a synergistic signal in response to ligands. As described in this study, the formation of a heterodimer between Dpp and Scw resolves these issues. In tissue culture assays, Scw homodimers produce very limited signal, while Dpp homodimers produce a moderate signal requiring only the Tkv receptor. The differential signaling ability of each homodimer explains their nonequivalence in producing patterning abnormalities when misexpressed during development. In contrast, the Dpp/Scw heterodimer is able to produce a synergistic phosphorylation of Mad that requires both the Tkv and Sax receptors; simply mixing homodimers is not sufficient. These observations demonstrate that synergistic signaling occurs at the level of receptor-mediated Mad phosphorylation and not through integration of separate signals at downstream targets. The molecular mechanism by which the Tkv and Sax receptors produce a synergistic output remains unclear (Shimmi, 2005).
Although the original role for Scw in dorsal patterning invoked formation of a heterodimer as the primary signaling species, this model fell into disfavor because ventral injection of scw mRNA or ventral expression of scw from the twist promoter can partially rescue amnioserosa formation. Since disulfide-linked heterodimer formation of TGF-β type ligands is known to occur in the Golgi during the secretion process, ventral expression of Scw without Dpp should preclude formation of heterodimers, and, therefore, any rescuing activity should be brought about by homodimers. Although some rescue was observed in these experiments, it is important to note that even multiple copies of ventrally expressed Scw do not lead to viability. In contrast, a single copy of Scw expressed in the dorsal domain using the tld promoter gives complete viability and fertility. In addition, these experiments assume that there is no internalization within the dorsal domain of Scw homodimers followed by isomerization with Dpp and resecretion. This possibility is mechanistically very similar to models in which Dpp is proposed to undergo transcytosis. Therefore, while ventral overexpression of Scw homodimers may have some ability to compensate for loss of Scw dorsally, normal patterning is most efficiently achieved when Scw is expressed in a domain in which heterodimers can form (Shimmi, 2005).
BMP-directed patterning of dorsal blastoderm cells ultimately results in the specification of two tissues, amnioserosa and dorsal ectoderm. In general, these tissues derive from cells receiving high and low BMP signal, respectively. Whether there are additional cell fate subdivisions specified within the steep signaling transition zone is not clear, although cells can discriminate subtle signaling differences as evidenced by the slightly wider expression pattern of the BMP target genes rho and usp compared to zen and race. Although both Dpp and Scw are required to establish the high point of signaling necessary to specify amnioserosa, only Dpp is needed to specify dorsal ectoderm. This is consistent with observations that the Dpp/Scw heterodimer will be preferentially concentrated at the midline because of its high affinity for Sog and Tsg. In contrast, Dpp and Scw homodimers will be more broadly distributed because of their lower affinities for Sog and Tsg. Although the different species cannot be directly distinguished in vivo, analysis of downstream target genes in a scw mutant embryo revealed that there is sufficient BMP activity to activate pnr transcription, but its pattern is very wide, consistent with the observed broad distribution of Dpp homodimers. In the wild-type case, Dpp and Scw homodimers, together with a small number of heterodimers that escape from Sog and Tsg, may contribute to signaling in the lateral ectoderm, since the pnr signal is stronger in wild-type than in scw mutants. These homodimers also likely signal in a repressive manner to prevent ectopic transcription of neurogenic genes within the dorsal domain. Thus, patterning of dorsal tissue appears to take advantage of the differing properties of homo- and hetero-dimers to establish a biphasic signaling state. Specifically, selective transport of the heterodimer and synergistic receptor signaling produce a restricted high point and amnioserosa cell fate, while Dpp and Scw homodimers generate a broad low level of signal that help fate the future dorsal ectoderm and restrict neurogenic activity to more lateral regions. It is likely that the full specification of dorsal ectoderm does not occur until a second round of dpp transcription takes place after germ band extension. It is also likely that additional components help reinforce the formation of the biphasic state, since recent genetic data indicate that tight localization of Dpp to the midline requires an initial phase of low-level Dpp signal reception (E.L. Ferguson, personal communication). The suggestion is that this initial low-level Dpp signal induces expression of an additional component that participates in the localization process. The identity of this component remains elusive (Shimmi, 2005).
Lastly, it is noted that employment of heterodimers in early embryonic patterning may be a common theme. In zebrafish, both BMP2b and BMP7 are required for dorsal-ventral patterning, and loss-of-function mutations in each gene exhibit identical severely dorsalized phenotypes. Since this phenotype is not enhanced in double mutants and overexpression of these two gene products reveals synergy in the ventralization of wild-type embryos, it has been suggested that BMP2a/BMP7 heterodimers are the primary molecules that specify ventral cell fates in this organism. These observations further highlight the overall similarity in the molecular components used to pattern the early zebrafish and Drosophila (Shimmi, 2005).
Use of the Dpp/Scw heterodimer provides the patterning system with an effective buffer at a very early step in dorsal cell fate specification. Buffering for reductions of Scw or Dpp is predominantly determined by the relative monomer production rates, and if Scw is in slight excess with respect to Dpp, reductions in the levels of Scw will have little effect on the output Dpp/Scw heterodimer, regardless of the specific choices of parameters (Shimmi, 2005).
Patterning is also resilient to reductions of Sog and Tsg. Sog and Tsg have synergistic BMP binding activity and the concentration of Sog/Tsg in the PV space is governed by the interaction of reaction and diffusion. The Sog/Tsg ratio can be computed as described for Dpp/Scw to determine the compensation in this subsystem, and the results are different from those for Dpp/Scw. Now there are two distinct solution regions, one for small β (β is the ratio of the wild-type production rates for monomer Dpp to monomer Scw) where many choices of parameters provide significant compensation for reductions of gene dosage, and one for large β where there is virtually no compensation. Because the behavior for large β and small β is very different, this analysis can explain the compensation for reductions in either Sog or Tsg but not both. This suggests that other mechanisms must be involved to explain the experimentally observed resilience in both sog and tsg heterozygous embryos. These could include the following: (1) the spatial separation of Sog and Tsg expression, (2) downstream kinetic mechanisms that compensate after Sog/Tsg formation, or (3) both. Both may contribute, but the following focuses on the possible effects of compensation in downstream kinetic interactions (Shimmi, 2005).
After Sog/Tsg formation, the next step downstream is the binding of the inhibitor Sog/Tsg to Dpp/Scw. Experimentally, it is observed that Tolloid cleavage of Sog is greatly enhanced when bound to Dpp/Scw and is enhanced in the presence of Tsg. In addition, a previous mathematical model of BMP patterning suggested that cleavage of Sog (only when bound in the complex Sog/BMP) is a requirement for the system to exhibit resilience to changes in gene dose of sog, tsg, or scw. These data support the idea that Dpp/Scw transported from the broad dorsal region must be released from the Sog/Tsg/Dpp/Scw complex. Interestingly, the local dynamics of Sog/Tsg + Dpp/Scw complex formation are completely analogous to the local dynamics for Sog + Tsg complex formation. This suggests that, if the level of Dpp/Scw or Sog/Tsg is decreased from the original wt levels, the output complex Sog/Tsg/Dpp/Scw would be less affected. Taken together, the Sog/Tsg and Sog/Tsg/Dpp/Scw steps lead to a cascade in which the compensation in the first step is enhanced in the second step. In effect, the output from one complex formation stage becomes the input substrate for the next stage. Of course, the level of buffering achieved depends on the system parameters. The output suggests that patterning would be most compensated for reduction of Scw, followed by Tsg, then Sog, and lastly Dpp. Of course, other downstream steps may also contribute to compensation (Shimmi, 2005).
In reality, patterning involves diffusive transport as well, but the analysis shows how a cascade of stages can produce compensation in the kinetic steps. When the full BMP patterning model that incorporates transport is compared to a previous model mediated by homodimers and monomers, there are approximately 100 times more 'robust' hits when scw+/−, sog+/−, tsg+/−, and tld+/− cases are considered. In principle, the binding cascade analysis extends to other systems and can be used to explore other changes of input, including overexpression of a protein (Shimmi, 2005).
In the Drosophila embryo, formation of a bone morphogenetic protein (BMP) morphogen gradient requires transport of a heterodimer of the BMPs Decapentaplegic (Dpp) and Screw (Scw) in a protein shuttling complex. Although the core components of the shuttling complex--Short Gastrulation (Sog) and Twisted Gastrulation (Tsg)--have been identified, key aspects of this shuttling system remain mechanistically unresolved. Recently, it was discovered that the extracellular matrix protein collagen IV is important for BMP gradient formation. This study formulates a molecular mechanism of BMP shuttling that is catalyzed by collagen IV. Dpp is shown to be the only BMP ligand in Drosophila that binds collagen IV. A collagen IV binding-deficient Dpp mutant signals at longer range in vivo, indicating that collagen IV functions to immobilize free Dpp in the embryo. In vivo evidence is provided that collagen IV functions as a scaffold to promote shuttling complex assembly in a multistep process. After binding of Dpp/Scw and Sog to collagen IV, protein interactions are remodeled, generating an intermediate complex in which Dpp/Scw-Sog is poised for release by Tsg through specific disruption of a collagen IV-Sog interaction. Because all components are evolutionarily conserved, it is proposed that regulation of BMP shuttling and immobilization through extracellular matrix interactions is widely used, both during development and in tissue homeostasis, to achieve a precise extracellular BMP distribution (Sawala, 2012).
There is ample experimental and theoretical support for the notion that BMP gradient formation in the early embryo involves the concentration of the most potent signaling species, the Dpp/Scw heterodimer, at the dorsal midline in a process involving Sog and Tsg. This study presents in vivo evidence for a role of collagen IV in two key aspects of this shuttling model, which have remained mechanistically unresolved. First, collagen IV functions to immobilize free Dpp, explaining why Sog and Tsg are needed for Dpp movement. Second, collagen IV acts as a scaffold for assembly of the Dpp/Scw-Sog-Tsg shuttling complex. The advantage to BMP gradient formation of assembling the shuttling complex on collagen IV has been suggested by analysis of organism-scale mathematical models. These models reveal that the in vitro binding affinity between BMPs and Sog is too low to account for the rate of shuttling complex formation required in vivo. However, by acting as a scaffold, collagen IV would increase complex formation by locally concentrating Dpp/Scw and Sog. Models with a 10–20% reduction in diffusion rates for Dpp/Scw and Sog and an increased apparent affinity of Dpp/Scw for Sog, show the best fit to in vivo data (Sawala, 2012).
The molecular model of shuttling complex assembly occurs in three steps. The first step involves independent binding of Dpp/Scw and Sog to collagen IV. The ability of Dpp-Δa to signal long range in sog− embryos, where wild-type Dpp is trapped in its expression stripe, provides in vivo evidence that the Dpp-collagen IV interaction restricts movement of free Dpp ligands. The result also demonstrates that Sog and Tsg promote long-range movement of Dpp because they release Dpp from collagen IV, and not simply because they prevent Dpp–receptor interactions. Restriction of Dpp diffusion by collagen IV may stabilize the gradient by preventing ventral movement of Dpp/Scw after release from Sog/Tsg and promoting Dpp/Scw–receptor interactions at the dorsal midline. It will be interesting, ultimately, to directly visualize Dpp and Dpp-Δa directly in sog and tsg mutant embryos. Although current methods allow detection of high levels of receptor-bound Dpp, there are technical limitations associated with specifically detecting the pools of Dpp that would be informative here, i.e., Dpp/Scw heterodimer within the shuttling complex or Dpp-Δa/Scw diffusing between cells. The data show that Scw is unable to bind the NC1 domain of collagen IV. This lack of collagen IV-dependent immobilization can explain why Scw, unlike Dpp, is capable of long-range signaling in the absence of Sog (Sawala, 2012).
Step 2 of shuttling complex assembly involves remodeling of the protein interactions to generate a poised intermediate. Specifically, step 2 is driven by Scw-mediated disruption of the Sog CR4–collagen IV interaction, so that Dpp/Scw is transferred from collagen IV to the Sog CR3-CR4 domains. Scw displacement of the Sog CR4 domain from collagen IV provides molecular insight as to why Scw is needed for Dpp transport. In addition to the binding preference of Sog and Tsg for the Dpp/Scw heterodimer, only Scw has a high affinity for the Sog CR4 domain. Therefore, Dpp/Scw can be released from collagen IV into the shuttling complex, whereas the Dpp homodimer remains trapped on collagen IV (Sawala, 2012).
In the final step of the model, Tsg mobilizes the shuttling complex by disrupting the Sog CR1–collagen IV interaction. It has been noted that tsg mutants display a more severe reduction in BMP signaling than sog and sog tsg double mutants. This observation has been attributed to a potential Sog-independent pro-BMP activity of Tsg at the level of receptor binding. A second contributing factor is suggested by the model, where Sog and Tsg act at distinct steps to allow formation of the shuttling complex. In tsg mutants, Dpp/Scw is loaded onto Sog by collagen IV, but remains locked in this inhibitory poised complex, so that the only BMPs capable of signaling are Dpp and Scw homodimers, which are less potent than the Dpp/Scw heterodimer. By contrast, in sog or sog tsg mutants, Dpp/Scw is not shuttled dorsally but is still capable of signaling locally, adding to signaling by Dpp and Scw homodimers. The weaker level of Dpp/Scw signaling in tsg mutants also provides support for the proposed order of steps 2 and 3 in the assembly process, because this order gives rise to the inhibitory intermediate of Dpp/Scw-Sog. Previously it was shown that an N-terminal fragment of Sog, called Supersog, which contains the CR1 domain and a portion of the stem, can partially rescue the loss of peak Dpp/Scw signaling in tsg− embryos. The model suggests that this property of Supersog comes from the ability of its CR1 domain to compete with full-length Sog for binding to collagen IV, thereby releasing Sog-Dpp/Scw, similar to the role of Tsg in shuttling complex assembly. It is noted that the CR1–collagen IV interaction appears weaker than that of CR4–collagen IV, which may facilitate release of Dpp/Scw by Tsg or Supersog-like fragments. After Tsg-mediated release from collagen IV, the mobile shuttling complex can diffuse randomly. Upon Tolloid cleavage of Sog, the liberated Dpp/Scw heterodimer rebinds collagen IV, which either promotes receptor binding or a further round of shuttling complex assembly, depending on the local concentration of Sog (Sawala, 2012).
In addition to collagen IV, the basic region in Dpp/BMP2/4 also binds to heparan sulfate proteoglycans (HSPGs), which can either restrict or enhance BMP long-range movement. Indeed, this study found that an HSPG-binding mutant, Dpp-ΔN, also binds only weakly to collagen IV, suggesting that the collagen IV- and HSPG-binding sites on Dpp overlap. It will be interesting to test how HSPGs and collagen IV interact to regulate BMP activity in tissues where they are coexpressed, such as the early vertebrate embryo. In the early Drosophila embryo, the absence of glycosaminoglycan chains, which largely mediate binding of HSPG to Dpp, make it possible to specifically focus on the Dpp–collagen IV interaction (Sawala, 2012).
A shuttling-based mechanism of BMP transport is also used in a number of other developmental contexts, including the early vertebrate embryo, specification of the vertebral field in mice, and establishment of the posterior cross-vein territory in the Drosophila wing disk. Restriction of BMP movement may also be important in other contexts, including several where collagen IV was already shown to regulate a short-range Dpp signal, such as the ovarian stem cell niche and the tip of malpighian tubules. The basic collagen IV binding motif is highly conserved among the Dpp/BMP2/4 subfamily and is also found in some other BMPs, including BMP3, consistent with reports that BMP3 and BMP4 can bind collagen IV. Overall, these findings support the idea that the collagen IV–BMP interaction is a conserved aspect of extracellular BMP regulation and suggest that the function of collagen IV in both long-range BMP shuttling and local restriction of BMP movement will impact on a number of other contexts in both flies and vertebrates (Sawala, 2012).
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