BIOLOGICAL OVERVIEW

The Screw protein shares a structural affinity with DPP since both are members of the TGF-ß superfamily. Their resemblence extends beyond structure: as null mutations in scw are phenotypically similar to moderate dpp mutants and cause dorsal cells to adopt ventral fates.

Although SCW mRNA is found ubiquitiously in early embryos, SCW activity is required only in dorsal cells. This has been demonstrated experimentally (Arora, 1994). A tolloid promoter was used to restrict scw transcription to dorsal cells. scw mutant flies expressing wild type screw only in the dorsal domain have a normal dorsal pattern, suggesting that SCW needs to function only in this region.

Excess DPP can overcome the requirement for scw function. scw mutants lack the amnioserosa, the dorsal most tissue layer of the developing fly. Following injection with DPP messenger RNA, 60% of scw null mutants differentiate a large number of amnioserosa cells. Thus, in embryos that lack scw, increased levels of DPP can suppress the scw mutant phenotype and restore the amnioserosa [Images]. Since DPP can function in the absence of scw, it is clear that dpp is not downstream of scw .

The simplest explanation for these results is that ubiquitously expressed scw somehow enhances the activity of DPP in dorsal cells where dpp is expressed. TGF-ß-like proteins in vertebrates (activins and inhibins) function as homodimers and heterodimers to produce divers biological effects, suggesting that DPP and SCW could likewise function as heterodimers, with the heterodimer producing a stronger biological effect than could be produced by DPP homodimers alone (Arora, 1994).

Although scw is expressed uniformly during blastoderm stages, its effects appear to be graded, and are restricted to the dorsal side of the embryo. DPP activity alone appears to be insufficient to specify different dorsal cell fates. SCW and DPP actiing together establish distinct response boundaries within the dorsal half of the embryo, perhaps by forming heterodimers that have higher activity than homodimers of either molecule acting alone (Arora, 1994).

Interpretation of a BMP activity gradient in Drosophila embryos depends on synergistic signaling by two type I receptors, SAX and TKV

In a dpp null mutant, all dorsal cell fates are missing and the embryos are completely ventralized. In contrast, embryos mutant for scw are partially ventralized and lack amnioserosa but differentiate a reduced dorsal ectoderm. The relative severity of the dpp and scw mutant phenotypes does not correlate with their expression patterns, since scw is transcribed uniformly at the syncitial blastoderm stage and dpp expression is restricted to the dorsal side of the embryo. One explanation for the different efficacies of the two ligands could be that they differ in abundance or have different affinities for their receptors. Alternatively, the ligands could evoke qualitatively different responses, perhaps by acting through different receptors. To distinguish between these alternatives, the ability of SCW mRNA to restore dorsal pattern in dpp null embryos was assayed. If the difference in the scw and dpp mutant phenotypes simply reflects their effective concentrations, excess Scw protein should compensate for the loss of dpp function. Injected Scw protein fails to restore amnioserosa in embryos that lack dpp function. This suggests that Scw and Dpp act in qualitatively distinct ways. While it had been postulated that dimerization between Scw and Dpp potentiates Dpp signaling by the formation of a potent Scw/Dpp dimer, this has been shown not to be the case. Expression of Scw in ventral cells in which Dpp is absent, rescues a scw mutant phenotype. Because Scw/Dpp dimers are likely to form intracellularly, these results strongly argue that formation of Scw/Dpp heterodimers is not a prerequisite for the biological activity of Scw in the embryo (Nguyen, 1998).

To understand the basis for the differential response of the embryo to Scw and Dpp signaling, the interaction of the ligands with the two type I receptors Sax and Tkv was examined. Using dominant-negative forms of the type I receptors Sax and Tkv, it is demonstrated that Sax mediates the Scw signal, while Tkv is required for both Dpp and Scw activity. While Dpp/Tkv signaling is obligatorily required, Scw/Sax activity is necessary but not sufficient for dorsal patterning. Tkv function is required for the response to both ligands, while the ability of Sax-DN to interfere specifically with Scw and not Dpp signaling strongly argues that Sax preferentially mediates the response to Scw. Sax and Tkv act synergistically, suggesting a mechanism for integration of the Scw and Dpp signals. Further, it is shown that the extracellular protein Sog can antagonize Scw, thus limiting its ability to augment Dpp signaling in a graded manner (Nguyen, 1998).

Genetic and phenotypic studies have established that sog and dpp exert opposing influences on dorsal patterning, leading to the suggestion that Sog functions as an antagonist of Dpp activity. Levels of Sog that do not affect Dpp signaling can block the ability of Scw to promote dorsal cell fates. The ability of Sog to specifically interfere with Scw does not conflict with previous studies showing a genetic antagonism of dpp activity by sog. Since Scw augments Dpp signaling, the inhibition of Scw activity by Sog is equivalent to antagonism of Dpp. In fact, results from earlier studies support the assertion that Sog preferentially targets Scw activity in the embryo. Thus, it is proposed that one way by which Sog mediates its negative effect on dorsal patterning is by antagonizing Scw function (Nguyen, 1998).

These data are also inconsistent with a central role for sog in modulating Dpp activity in late development. Ectopic expression of Sog in the wing disc using a variety of GAL4 drivers causes no significant phenotypic defects. This is quite striking given the prominent role of Dpp in organizing pattern along the anterior-posterior axis in the wing disc. It is worth noting that the loss of posterior crossveins caused by expression of Sog is similar to the defect caused by Sax-DN, rather than Tkv-DN. An explanation for the failure of Sog to target Dpp could be that Dpp is bound to extracellular matrix components or forms a high-affinity complex with its receptor. Alternatively, the observation that Xenopus Noggin can severely ventralize Drosophila embryos raises the possibility that a Noggin-like factor may be the functionally relevant Dpp antagonist (Nguyen, 1998).

If Sog primarily blocks Scw activity during embryogenesis, the role of Tolloid may be to potentiate Scw signaling by releasing it from an inhibitory complex. Scw can promote Tld-dependent cleavage of Sog. This may explain why the loss of tld function results in a partially ventralized phenotype similar to that of scw- mutants, rather than the complete ventralization typical of dpp null embryos. The observation that embryos lacking both scw and tld function do not display a more severe phenotype is also compatible with this view (Nguyen, 1998).

Different requirements for proteolytic processing of bone morphogenetic protein 5/6/7/8 ligands in Drosophila melanogaster

Bone morphogenetic proteins (BMPs) are synthesized as proproteins that undergo proteolytic processing by furin/subtilisin proprotein convertases to release the active ligand. This study examined processing of BMP5/6/7/8 proteins, including the Drosophila orthologs Glass Bottom Boat (Gbb) and Screw (Scw) and human BMP7. Gbb and Scw have three functional furin/subtilisin proprotein convertase cleavage sites; two between the prodomain and ligand domain, which are called the Main and Shadow sites, and one within the prodomain, which is called the Pro site. In Gbb each site can be cleaved independently, although efficient cleavage at the Shadow site requires cleavage at the Main site, and remarkably, none of the sites is essential for Gbb function. Rather, Gbb must be processed at either the Pro or Main site to produce a functional ligand. Like Gbb, the Pro and Main sites in Scw can be cleaved independently, but cleavage at the Shadow site is dependent on cleavage at the Main site. However, both Pro and Main sites are essential for Scw function. Thus, Gbb and Scw have different processing requirements. The BMP7 ligand rescues gbb mutants in Drosophila, but full-length BMP7 cannot, showing that functional differences in the prodomain limit the BMP7 activity in flies. Furthermore, unlike Gbb, cleavage-resistant BMP7, although non-functional in rescue assays, activates the downstream signaling cascade and thus retains some functionality. These data show that cleavage requirements evolve rapidly, supporting the notion that changes in post-translational processing are used to create functional diversity between BMPs within and between species (Fritsch, 2012).

While the results of this study support the conventional dogma that proteolytic processing and dissociation of the prodomain are essential steps in BMP activation, this study shows that different BMPs, even if closely related, can have distinct processing requirements. In particular, for the BMP5/6/7/8 proteins, a novel mechanism of prodomain shedding was identified involving Furin-mediated cleavage at a site within the prodomain, and evidence is provided that some BMPs can signal with part or all of the prodomain covalently attached (Fritsch, 2012).

Gbb processing occurs at three sites to give rise to an N-terminal pro-fragment, a C-terminal profragment, and the ligand domain. The processing event that must occur to activate Gbb is the separation of the ligand domain from the N-terminal pro-fragment, which can occur by cleavage at either the Pro site or the Main site. Thus, Gbb can function either as a fully processed ligand or with the C-terminal fragment of the prodomain covalently attached. An inhibitory role for the N-terminal part of the prodomain is not without precedent: functional studies on Myostatin/GDF8 have mapped the inhibitory domain of the protein to amino acids 42 to 115, and the recent crystal structure of TGF-β1 has also implicated this part of the protein in blocking ligand function (Fritsch, 2012).

Scw is processed at three cleavage sites orthologous to those in Gbb (Pro, Main and Shadow), but in this case, cleavage at both the Pro site and the Main site are required to produce a functional ligand. Cleavage at the Pro site is required for dissociation of the prodomain and for stable accumulation of the secreted ligand. Thus, when complexed with the prodomain, Scw is either inefficiently secreted or rapidly internalised following secretion, which is similar to what has been reported for members of the BMP2/4/Dpp group. Cleavage at the Main site is required for dissociation of the ligand domain from the Cterminal prodomain fragment, which, in Scw, inhibits ligand function. The inhibitory effect of the C-terminal prodomain region in Scw is a recent evolutionary adaptation, and could reflect either a newly evolved property of the prodomain or a newly evolved function of the Scw ligand domain with which this prodomain fragment interferes. Notably, a similar rapid functional divergence in the prodomain has also been reported for BMP15, where the prodomains of the mouse and human orthologs confer differential processing efficiencies to the pro-protein that may underlie the distinct functional properties of the ligand in these two species (Fritsch, 2012).

The data support the model that human BMP7 is processed at a single, low-probability Furin cleavage site that lies between the prodomain and ligand domain, and that cleavage at this site is essential for function. Curiously, while the Gbb-BMP7 ligand domain chimera fully rescues gbb mutants, full length BMP7 does not, suggesting that the BMP7 prodomain is incompatible with essential features of Gbb processing and signalling. Thus, while the Gbb and BMP7 ligands are functionally interchangeable, their pro-domains have diverged, presumably to fine-tune their activity to fulfil their endogenous functions (Fritsch, 2012).

Taken together these findings on Scw, Gbb and BMP7 illustrate how evolutionary plasticity in the prodomain sequence serves to modulate the activity of the ligand, which may be subject to stronger evolutionary constraints and thus unable to diverge sufficiently to provide functional diversity. The Furin-mediated cleavage of Gbb and Scw characterized in this study is one of a number of different prodomain processing events that have been shown to modulate the function of TGFβ superfamily ligands. For many ligands, the prodomain has been shown to remain noncovalently associated with the ligand, and a range of different functions have been ascribed to it, including targeting the complex for degradation or to the extracellular matrix. Indeed, the prodomains of BMP-4, -5, -7, -10 and GDF5 have been shown to interact with Fibrillin, and the GDF8 prodomain with Perlecan, and various strategies are deployed to dissociate this complex and release the active ligand. In vitro evidence suggests that for BMP7, the prodomain can be displaced by the Type II receptor. A more general strategy may be proteolytic cleavage of the prodomain. GDF11, GDF8 and BMP10 require proteolytic cleavage by the metalloproteinase Tolloid/BMP-1 to activate signalling in vivo or in tissue culture assays. The data suggest that an additional mechanism for activation is Furin/SPC cleavage of the prodomain (Fritsch, 2012).

The recent crystal structure of the pro-TGF-β1 dimer reveals that the two prodomains form a ring-like shape that wraps around the ligand dimer, altering its structure and shielding it from interaction with receptors. The prodomain ring is subdivided into C-terminal 'arms', an extended loop that encircles the tip of each ligand monomer, and two N-terminal, α-helical forearms that cross one another forming a ‘straitjacket’ around the ligand. In the TGF-β1 model, the N-terminal straitjacket locks the ligand dimer into the prodomain ring, and mechanical force induced by interaction with extracellular matrix proteins unfastens the straitjacket to release the active ligand (Fritsch, 2012).

This model for the prodomain fold provides insight into the molecular basis for results on the processing requirements for Gbb, Scw and BMP7, and also raises intriguing questions. The Pro site in Gbb and Scw lies within the extended loop between the arm and straitjacket, and thus is in a key position to influence ligand accessibility and function. In the case of Gbb, cleavage at this site effectively opens the straitjacket and reveals the ligand dimer irrespective of cleavage at the Main/Shadow sites. Scw, on the other hand, cannot function even with the straitjacket removed in this way. This distinction suggests there is a difference between the arm domains of Gbb and Scw, the former of which is neutral to ligand function and the latter of which is inhibitory. Moreover, Scw requires cleavage at the Pro site to shed the prodomain and activate the ligand, while Gbb functions without this cleavage. Thus, Scw follows the TGF-β1 paradigm of requiring more than just cleavage at the Main site to shed the prodomain, while Gbb presents a distinct situation where cleavage at the Main site is sufficient for prodomain shedding (Fritsch, 2012).

The results with BMP7 also depart from the TGF-β1 paradigm where the prodomain locks the ligand into an inactive form. While cleavage-resistant BMP7 only retains a low level of signalling activity, the data show that it can activate the signalling pathway despite having the prodomain covalently attached. While the ability for a BMP pro-protein to bind to its receptor has been previously shown for BMP2 and BMP7 in vitro, the data provide the first evidence that a BMP pro-protein can signal in vivo without displacement of the prodomain (Fritsch, 2012).

The expression of cleavage-resistant TGFβ superfamily proteins has been shown to generate dominant negative effects by blocking secretion of the wild type protein. In these studies, it was assumed that the mutant proteins formed heterodimers with the wild type monomers, thus promoting their degradation within the cell. The results with cleavage resistant Gbb do not support this type of model for two reasons. First, cleavage resistant Gbb is secreted at normal levels, and thus is not degraded within the cell. Second, it was found that cleavage-resistant Gbb knocks down the function of endogenous Gbb, but not Dpp, with which it forms heterodimers. Thus, the dominant negative effect is exclusive to the homotypic ligand. This specificity has been reported previously, but it is not clear how it might arise for a protein that is known to function by forming heterodimers with other ligands. This suggests that either the relationship between dimerization and processing is different than currently thought, or that the mechanism underlying the dominant negative behaviour is not exclusively due to heterodimerization (Fritsch, 2012).

Phylogenetic analysis of the BMP5/6/7/8 proteins has shown that the processing sites are embedded in blocks of sequence that are poorly conserved even between closely related species. This feature is also a characteristic of the cleavage sites in the BMP2/4/Dpp proteins, and thus the domains that include the cleavage sites in the BMPs appear to be in constant flux, with the core and flanking sequences changing from one species to the next. Indeed, in both subgroups there is evidence that the arrangement of cleavage sites can change, presumably influencing the processing mechanism. For example, in the vertebrate BMP2/4 proteins, the S1 site is a high probability site and is cleaved first, while the S2 site is a low probability site and is only cleaved after processing at the S1 site. In Arthropods, these cleavage probabilities are reversed (and an additional cleavage site is added) and the order of cleavage is correspondingly inverted. Similarly, in the Gbb proteins, the Main site is typically a high probability site, and the Shadow site a low probability site, but in Glossina, Anopheles, and Tribolium, the cleavage probabilities are reversed, indicating that, in Gbb, it is the presence, and not the position of the site that is important. In this light, the Furin cleavage sites appear to evolve like transcription factor binding sites in a promoter where the key feature is maintaining the function of the element irrespective of the position, number, or affinity of the binding sites that comprise it. Given this, the plasticity in processing requirements the observations for the BMP5/6/7/8 proteins may well apply to other BMPs and be a general mechanism whereby the ligands are fine-tuned for their particular functions (Fritsch, 2012).

GENE STRUCTURE

The screw gene is found in the midst of an intron of 90 kb long Dlar gene

Bases in 5' UTR - 114

Exons - 1

Bases in 3' UTR - 99 and 405 corresponding to the two transcripts

PROTEIN STRUCTURE

Amino Acids - 400

Structural Domains and Evolutionary Homologies

The SCW protein has extensive homology to members of the TGF-ß superfamily. Proteins of this class are typically synthsized as inactive dimers that undergo proteolytic cleavage to generate a mature carboxy-terminal segment that forms the ligand molecule. The SCW precursor contains an amino-terminal signal sequence and several amino linked glycosylation sites. The conserved carboxy-terminal region is immediately preceded by a series of basic residues that could form a site for proteolytic cleavage of the precursor protein. A distinguishing feature of TGF-ß proteins is the presence of seven invariant cysteine residues in the carboxy-terminal region of the molecules. SCW has 40% identity with DPP in the carboxy-terminal region. However DPP exhibits a closer relationship to vertebrate BMP-2 and BMP-4 than does SCW.

Transcriptional Regulation

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).

Protein Interactions

Given the structural similarity between DPP and SCW, it is likely that SCW functions as a signaling molecule by forming heterodimers with DPP. It is thought that DPP homodimers are capable of signaling, but that SCW/DPP heterodimers elicit a stronger response (Arora, 1994).

Genetic interaction between certain scw and dpp alleles is consistent with a physical association of the proteins encoded by these genes. Embryos carrying a single copy of a partial loss-of function dpp allele and a single copy of a specific mutant allele of scw (trans-heterozygotes) die with a partially ventralized phenotype. In contrast, embryos that are homozygous for the scw mutation and carry a single copy of the dpp mutant allele are completely viable, indicating that the defective product encoded by the scw allele can block the activity of the single functional copy of dpp. The defective SCW proteins may be incapable of signal transduction but still are able to sequester active DPP molecules and thus reduce the effective levels of DPP (Raftery, 1995).

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).

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

Patterning the dorsal surface of the Drosophila blastoderm embryo requires Decapentaplegic (Dpp) and Screw (Scw), two BMP family members. Signaling by these ligands is regulated at the extracellular level by the BMP binding proteins Sog and Tsg. 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).

Multistep molecular mechanism for bone morphogenetic protein extracellular transport in the Drosophila embryo

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).

Adaptive protein divergence of BMP ligands takes place under developmental and evolutionary constraints

The bone morphogenetic protein (BMP) signaling network, comprising evolutionary conserved BMP2/4/Decapentaplegic (Dpp) and Chordin/Short gastrulation (Sog), is widely utilized for dorsal-ventral (DV) patterning during animal development. A similar network is required for posterior crossvein (PCV) formation in the Drosophila pupal wing. Although both transcriptional and post-transcriptional regulation of co-factors in the network appears to give rise to tissue-specific and species-specific properties, their mechanisms are incompletely understood. In Drosophila, BMP5-8 type ligands, Screw (Scw) and /aGlass bottom boat (Gbb), form heterodimers with Dpp for DV patterning and PCV development, respectively. Sequence analysis indicates that the Scw ligand contains two N-glycosylation motifs; one being highly conserved between BMP2/4 and BMP5-8 type ligands, and the other being Scw ligand-specific. The data reveal that N-glycosylation of the Scw ligand boosts BMP signaling both in cell culture and in the embryo. In contrast, N-glycosylation modifications of Gbb or Scw ligands reduce the consistency of PCV development. These results suggest that tolerance for structural changes of BMP5-8 type ligands is dependent on developmental constraints. Furthermore, gain and loss of N-glycosylation motifs in conserved signaling molecules under evolutionary constraints appear to constitute flexible modules to adapt to developmental processes (Tauscher, 2016).

This study provides insights into how evolutionary and developmental pressures shape molecules after their divergence from a common ancestor. A conserved N-glycosylation motif exists, which is specific for BMP-type ligands throughout various animal species. In addition, it was observed that the BMP5-8-type ligand Scw contains a unique N-glycosylation motif that helps to maintain a peak level of BMP signal in the embryo. In contrast, N-glycosylation modifications of BMP-type ligands reduce the consistency in PCV development. These observations provide insights into how evolutionarily conserved signaling molecules adapt to developmental processes (Tauscher, 2016).

The significance of N-glycosylation of the TGF-β-type ligands has been studied previously. For example, N-glycosylation of the BMP2 prodomain affects the folding and secretion of ligands, and non-glycosylated BMP2 and BMP6 produced in bacterial cells appear to be less active than the glycosylated ligands. Addition of an N-glycosylation motif in Nodal changes the stability of ligands, resulting in an increased signaling range. These facts suggest that N-glycosylation of ligands may play significant roles in vivo. However, these roles have been largely unexplored because of a lack of in vivo model systems. By employing both in vivo studies and cell-based experiments, this study has investigated how N-glycosylation modifications of the BMP-type ligands impact developmental processes. The in vivo rescue experiments revealed that these motifs are crucial for fly viability and are required to achieve peak level BMP signaling. Loss of the Scw-specific motif leads to a reduced impact on BMP signaling in the embryo compared with the effect of the conserved motif but also to less signaling capacity when compared to ScwWT, resulting in lower viability of g.scwN1Q rescued flies. On the other hand, integration of the Scw-specific N-glycosylation motif into its paralog Gbb (Scw-Gbb chimera) is not sufficient to provide functionality in the early embryo. This suggests that the critical changes responsible for the differing specificity of the Gbb and Scw ligands that developed after gene duplication may be differences in the primary sequences other than N-glycosylation motifs (Tauscher, 2016).

As reported in the case of Nodal, adding N-glycosylation sites to ligands may change protein stability/secretion and therefore may affect in vivo phenotypes. In the case of Scw, it is presumed that acquisition of the unique N-glycosylation motif has no drastic effect on protein stability/secretion, but instead directly affects the signaling outcome. First, equal amounts of differentially glycosylated ligands show different signaling intensities in the cell-based assay. Second, expression of differentially glycosylated ligands showed different signaling intensities in the embryo when they are expressed in identical genetic backgrounds. Third, the total protein levels in both cell lysates and supernatants for ScwWT, ScwN1Q and ScwN2Q are equivalent when they are expressed in S2 cells. Thus, these results suggest that changing the number and positions of N-glycosylation motifs may impact signaling intensities both in vivo and in vitro without significantly changing protein stability/secretion. In contrast, non-glycosylated Scw ligand (ScwN1_N2Q) appears to be less efficiently secreted. These facts suggest that at least one N-glycosylation site of Scw is crucial for maintaining protein stability/secretion, but their number or position may not be essential for secretion (Tauscher, 2016).

Interestingly, N-glycosylation of the ligands did not provide any advantage for PCV formation. Instead, the Scw ligand lacking both N-glycosylation motifs (ScwN1_N2Q) most efficiently restored the PCV-less phenotypes in gbb mutant wings. It is hypothesized that N-glycosylation of BMP ligands does not always benefit extracellular trafficking of ligands. Highly glycosylated ligands may interact with enriched extracellular matrix (ECM) at the basal side of wing epithelia and reduce the ligand mobility regulated by the BMP network. Alternatively, differential expression of key molecules may explain different phenotypes between embryogenesis and crossvein development. It has been previously reported that the heparan sulfate proteoglycan (HSPG) Dally impacts BMP signaling in various contexts. Dally plays a role in Dpp gradient formation in the wing imaginal disc by stabilizing Dpp and it increases the signaling of Gbb and Dpp in Drosophila S2 cells. In addition, lack of Dally and Dally-like protein (Dlp) affects PCV formation in the wing. Interestingly, HSPGs are absent within the first 3 hours of embryogenesis, which is the only time frame of scw expression. Based on these facts, it appears that Scw and HSPGs are mutually exclusive. This may partly explain why non-glycosylated Scw is functional for PCV development but not for embryonic DV patterning. Furthermore, the ScwN1_N2Q:Dpp heterodimer is likely to be a primary ligand responsible for BMP signaling in the PCV region. Since Dpp carries the conserved N-glycosylation motif, the ScwN1_N2Q:Dpp heterodimer contains one N-glycosylation site, although ScwN1_N2Q lacks N-glycosylation site. The N-glycosylation site of Dpp may help facilitate ScwN1_N2Q:Dpp heterodimer secretion (Tauscher, 2016).

Why is a unique N-glycosylation site acquired in the Scw ligand? scw is exclusively expressed in the early embryo, which is in contrast to the usually recurrent activity of signaling molecules at different stages of development. The favored model is that random mutations create differential N-glycosylation motifs in otherwise functionally redundant and conserved ligands. These novel motifs lead to structural changes that confer either advantages or disadvantages, depending on the developmental context. Since a positive feedback mechanism is crucial for DV patterning in Drosophila, acquisition of the unique N-glycosylation site could bring an advantage to Scw signaling. In contrast, in a wide range of species including humans, BMP2/4- and BMP5-8-type ligands are repeatedly utilized for development at different stages and in different positions. Therefore, to provide robustness and reproducibility in various contexts, vertebrate BMP2/4 and BMP5-8 contain only one N-glycosylation site to impose developmental constraints: stronger signaling than a non-glycosylated ligand, and less impeded extracellular trafficking than additionally glycosylated ligands. Consistently, Gbb has been shown to function at various developmental stages (Tauscher, 2016).

Although various co-factors of the BMP network have been identified among species, it remains to be addressed how they adapted to different developmental stages and different species. The scw allele was originally identified as a DV patterning defect and was determined to encode a BMP5-8-type protein. It was then proposed that scw originates from gene duplication of gbb in the branch leading to higher Diptera, a highly diverged branch in the arthropod lineage. Hence, gbb and scw provide an outstanding opportunity to investigate evolutionary divergence of protein structures. In Drosophila, gbb and scw are expressed in distinct patterns, but both function as co-factors of the BMP network. A recent study indicates both Gbb and Scw are utilized for DV patterning in the scuttle fly. gbb expression was also described in the early embryo of the lower Dipteran Clogmia albipunctata, in which the scw gene was not found. These facts indicate a possibility that Gbb acts as a co-factor of the BMP network for DV patterning in most arthropod species and that Scw evolved specifically for DV patterning in higher Diptera after duplication of the scw-like gene gbb. Further studies are needed to elucidate how Gbb lost the capacity to transduce signals in the Drosophila blastoderm embryo (Tauscher, 2016).

In summary, these data reveal that two BMP5-8-type ligands, Scw and Gbb, which function as co-factors of the BMP network, provide a unique model to investigate how orthologous proteins evolve under developmental and evolutionary constraints. Further studies in this context will help elucidate how evolutionarily conserved molecules generate diversified structures in the animal kingdom (Tauscher, 2016).

DEVELOPMENTAL BIOLOGY

Embryonic

scw gene is ubiquitously expressed during early embryogenesis. Moderate levels of SCW mRNA are first detected in stage 4 embryos toward the end of nuclear cycle 10, and increase rapidly. But levels soon decline and by the cellular blastoderm stage 5 [Image], SCW message has declined below detectable levels (Arora, 1994).

Advances in image acquisition and informatics technology have led to organism-scale spatiotemporal atlases of gene expression and protein distributions. To maximize the utility of this information for the study of developmental processes, a new generation of mathematical models is needed for discovery and hypothesis testing. A data-driven, geometrically accurate model has been developed of early Drosophila embryonic bone morphogenetic protein (BMP)-mediated patterning. Nine different mechanisms for signal transduction with feedback, eight combinations of geometry and gene expression prepatterns, and two scale-invariance mechanisms were tested for their ability to reproduce proper BMP signaling output in wild-type and mutant embryos. It was found that a model based on positive feedback of a secreted BMP-binding protein, coupled with the experimentally measured embryo geometry, provides the best agreement with population mean image data. The results demonstrate that using bioimages to build and optimize a three-dimensional model provides significant insights into mechanisms that guide tissue patterning (Umulis, 2010).

In many systems, spatially patterned cellular differentiation is regulated by signaling molecules called morphogens, which initiate spatiotemporal patterns of gene expression in a concentration-dependant manner. In early Drosophila embryos, a morphogen composed of a heterodimer of Decapentaplegic (Dpp) and Screw (Scw), two members of the bone morphogenetic protein (BMP) family. Unlike classical morphogen systems that rely on the slow spreading of a molecule from a localized source to establish a gradient, BMPs in the early Drosophila embryo are secreted from a broad region making up the dorsal-most 40% of the embryo circumference. Subsequently, they are dynamically concentrated into a narrow region centered about the dorsal midline that makes up only 10% of the embryo circumference (Umulis, 2010).

A number of extracellular regulators contribute to the dynamics and localization of BMP signaling. Laterally secreted Short gastrulation (Sog) and dorsally secreted Twisted gastrulation (Tsg) diffuse from their regions of expression and form a heterodimer inhibitor (Sog/Tsg) that binds to Dpp-Scw, preventing it from binding to receptors. The cell matrix may mediate the formation of this complex, as it has recently been shown that collagen can bind both BMPs and Sog, thereby facilitating their association (Wang, 2008). The extracellular binding reactions lead to a gradient of inhibitor-bound Dpp-Scw that is high laterally and low at the dorsal midline, and an opposing gradient of free Dpp-Scw that is high at the dorsal midline. The dorsally secreted metalloprotease Tolloid (Tld) processes Sog only when Sog is bound to BMP ligands, and the degradation of Sog by Tld further enhances both the gradient of inhibitor-bound Dpp-Scw and of free Dpp-Scw. Thus, extracellular Dpp-Scw is redistributed by a combination of binding to inhibitor, processing of this complex, and diffusion (Umulis, 2010).

Simultaneously, receptors and other surface-localized binding proteins compete with Sog to bind the available Dpp-Scw. Dpp-Scw activates signaling by binding to and recruiting the Drosophila type I receptors, Thickveins (Tkv) and Saxophone (Sax), into a high-order complex containing two subunits of the type II receptor Punt. The receptor complex phosphorylates Mad (pMad), a member of the Smad family of signal transducers, and phosphorlyated Mad binds to the co-Smad Medea, forming a complex that then accumulates in the nucleus, where it regulates gene expression in a concentration-dependent manner (Umulis, 2010).

Although complex formation and transport favor a net movement of ligand toward the dorsal midline of the embryo, positive feedback in response to pMad signaling is needed to further concentrate the surface-localized Dpp-Scw at the dorsal midline. A loss of extracellular BMP regulators or positive feedback impedes the attenuation of pMad laterally as well as the accumulation of pMad signaling at the dorsal midline. Although feedback, extracellular transport, and signal transduction each provide a specific mode of Dpp-Scw signal regulation, it is the dynamic interaction of these regulatory mechanisms that patterns the dorsal surface of Drosophila embryos. Not only does the mechanism work under optimal laboratory conditions, but dorsal surface patterning appears to be remarkably resilient to nonideal conditions such as temperature fluctuations, reductions in the level of regulatory factors such as Tsg, ectopic gene expression, and other perturbations. These issues illustrate the complexity of the problem and suggest that it is not possilbe to rely solely on genetic and biochemical data to fully explain this rather simple patterning problem (Umulis, 2010).

To address a number of unanswered questions about Dpp-Scw-mediated patterning and to take full advantage of the available data on Drosophila development, a methodology was developed that seamlessly integrates biological information in the form of prepatterns, geometry, mechanisms, and training data into an organism-scale model of the blastoderm embryo that is based on a reaction-diffusion description of patterning. The mathematical model is simulated by using the widely available computational frameworks Comsol and Matlab, which makes extensive use of the model and methodology feasible (Umulis, 2010).

An image analysis protocol was developed to obtain model training and initial condition data and to calculate population statistics for patterns of pMad signaling in wild-type (wt) and mutant D. melanogaster. Both the mean and variability of pMad signaling along the dorsal-ventral (DV) axis depends on anterior-posterior (AP) position and the specific choice of threshold. Using mutations previously considered robust, differences could be detected between mutant and wild-type pMad signaling patterns, which provided an information-rich data set for model training and for testing the contributions of diverse positive-feedback mechanisms and of proteins that concentrate BMPs at the cell surface. Unexpectedly, it was found that geometry also has a large impact on the predicted patterns of BMP-bound receptors, whereas the prepatterned expression of receptors and other modulators of signaling did not greatly affect model-data correspondence. It was found that if the embryo geometry is perturbed slightly in the model, then including the prepattern information greatly enhanced the model's ability to fit the observed pMad patterns, which suggests that the prepatterns may mitigate the effects of slightly misshapen embryos. Conditions in the model were identified that improve the scale invariance of patterning and tested the model predictions by staining for pMad in different species of Drosophila. These studies demonstrate that building a model based on image data and training the three-dimensional (3D) model against multidimensional expression data provide insights into the properties of several important developmental principles, including positive feedback, biological robustness, and scale invariance (Umulis, 2010).

Effects of Mutation or Deletion

Dorsal-ventral patterning within the embryonic ectoderm of Drosophila requires two type I TGFbeta receptors, Tkv and Sax, as well as two TGFbeta ligands, Dpp and Scw. In embryos lacking dpp signaling, increasing the level of Tkv activity promotes progressively more dorsal cell types, while activation of Sax alone has no phenotypic consequences. However, Sax activity synergizes with Tkv activity to promote dorsal development. To determine the interrelationship between the signaling pathways downstream of the Tkv and Sax receptors, an assay was carried out of the phenotypic consequences of activating each signaling pathway separately in embryos that lack dpp expression. Increasing levels of activation of Tkv signaling recapitulate embryonic dorsal-ventral pattern, as measured by the dosage-dependent production of dorsal epidermal and amnioserosal cell fates. In contrast, activation of the Sax signaling pathway alone does not promote formation of any dorsal structures. However, the activated Sax receptor synergizes with the activated Tkv receptor in production of both dorsal epidermis and amnioserosal cell fates. From these data it is concluded that, while the functions of both receptors are necessary for in vivo patterning, elevation of Tkv signaling can bypass the requirement for Sax signaling. Furthermore, the data indicate that Sax signaling is dependent on Tkv signaling for phenotypic consequences and that Sax signaling elevates the biological response to a given level of Tkv signaling (Neul, 1998).

Functional experiments suggest the two receptors have different ligands: Dpp acts through Tkv, and Scw acts through Sax. Furthermore, Sog, a negative regulator of this patterning process, preferentially blocks Scw activity. To establish functional interactions between the Scw ligand and the Sax receptor, use was made of the ability of scw mutant embryos to produce amnioserosa in response to injection of either DPP or SCW mRNAs. Injection of mRNA encoding a dominant-negative Sax receptor is able to block the biological activity of injected SCW mRNA but is unable to block the activity of injected DPP mRNA. These findings were extended by showing that scw function is required for the ability of a chimeric receptor containing the extracellular domain of Sax fused to the intracellular domain of Tkv to rescue a tkv mutant. Taken together, these results strongly suggest that Scw is an obligate component of the Sax ligand. Furthermore, because ventral expression of scw in cells that do not express dpp is sufficient to rescue a scw mutant, Scw-DPP heterodimers appear not to be essential for the generation of wild-type pattern, raising the possibility that Scw homodimers are the in vivo ligand for the Sax receptor (Neul, 1998).

Injection of SOG mRNA blocks the biological response of scw mutants to injection of SCW mRNA, but not to injection of DPP mRNA. These results strongly suggest that Sog, which has been genetically characterized as a negative regulator of Dpp activity, functions primarily to modulate Scw activity over the dorsal-ventral axis. These data thus suggest that an activity gradient of dpp results from the differential spatial modulation of Scw activity by Sog. This could happen by either of two mechanisms. One possibility is that the existence of a local ventral source for Sog and the presence of a 'sink' for Sog in the dorsal regions of the embryo (the cleavage of Sog by Tld) could result in a ventral-to-dorsal gradient of Sog. The binding of Sog to Scw could thereby result in the formation of a reciprocal dorsal-to-ventral gradient of scw activity. A second model for the action of Sog posits that Sog facilitates the directional diffusion of the Scw ligand from the lateral to the dorsal regions of the embryo. Specifically, Sog binding to Scw shields the ligand from binding to its ubiquitously localized receptors and thereby allows the Scw-Sog complex to diffuse in the perivitelline space. Dorsally located Tolloid then cleaves Sog, releasing the Scw ligand from the inhibitor. The action of Sog would thus lead to increased dorsal localization of Scw and increased activity of the Sax pathway, ultimately resulting in formation of amnioserosa. This facilitated diffusion model implies that one function of Sog is to elevate Dpp/Scw signaling dorsally. This model would directly explain the reduction in amnioserosa observed in sog mutants and would account for the cell nonautonomous function of Scw, revealed by ventral injections of SCW mRNA. Moreover, this model could also provide an explanation for a puzzling aspect of the phenotype of embryos that lack the nuclear gradient of dorsal gene product. Such dorsalized embryos have a pattern of zygotic gene expression around the embryonic circumference that is similar to that of the most dorsal cells in the wild-type embryo. However, only a small number of cells in dorsalized embryos differentiate as amnioserosa; the great majority of cells in these embryos differentiate as dorsal ectoderm. An increase in dpp gene dosage in dorsalized embryos is sufficient to increase the number of amnioserosal cells. Thus, it appears that despite the pattern of gene expression in dorsalized embryos, the level of dpp/scw signaling is not sufficient to fate amnioserosa. Dorsalized embryos do not express sog; thus, the lack of 'facilitated diffusion' of the Scw ligand mediated by Sog could be the cause of this phenotype (Neul, 1998 and references).

It is proposed that the original function of Dpp might have been to mediate dose-independent cell fate decisions. The ability of Dpp to function in a dose-dependent manner was acquired evolutionarily by the recruitment of a second signaling system whose output could modulate Tkv activity, but whose biological function was dependent on Dpp. The genetic compartmentalization inherent within this circuitry would have ensured the increased evolutionary capacity of such a patterning system. Specifically, genetic alterations in components of the modulatory signaling pathway could lead to significant phenotypic variability without disruption of the original cell fate choice mediated by Dpp. Thus, this genetic circuitry could have been a component in the generation of diverse body plans (Neul, 1998).

Mutations at five loci delete specific pattern elements in the dorsal half of the embryo and cause partial ventralization. Mutations in the genes zerknüllt and shrew affect cell division only in the dorsalmost cells corresponding to the amnioserosa, while the genes tolloid, screw and decapentaplegic affect divisions in both the prospective amnioserosa and the dorsal epidermis. In each of these mutants dorsally placed mitotic domains are absent and this effect is correlated with an expansion and dorsal shift in the position of more ventral domains. Null alleles of tolloid and screw are marked by an absence of the amnioserosa [Image] and a reduction of the dorsal cuticle accompanied by a 30% increse in the width of the ventral setal belts in each segment. Maxillary sense organs, that derive from a dorsolateral position on the blastoderm fate map, are absent, and antennal sense organs which arise from adjacent cells, are encountered only in a third of the mutant embryos. There is a loss of pharyngeal skeletal elements. The filzkörper, paired dorsolaterally derived structures in the tail of the larvae, are reduced or absent, and the thorax is characteristically coiled, hence the gene name screw (Arora, 1992).

The homeobox gene tinman plays a key role in the specification of Drosophila heart progenitors and the visceral mesoderm of the midgut, both of which arise at defined positions within dorsal areas of the mesoderm. In addition to the heart and midgut visceral mesoderm, tinman is also required for the specification of all dorsal body wall muscles. Thus it appears that the precursors of the heart, visceral musculature, and dorsal somatic muscles are all specified within the same broad domain of dorsal mesodermal tinman expression. Because of the crucial role of dpp in inducing dorsal mesodermal tinman expression and the specification of dorsal mesodermal tissues, it is of interest to determine whether other components known to function in dpp-mediated signaling events during blastoderm are also required for mesoderm induction. Screw, a second BMP2/4-related gene product, Tolloid, a BMP1-related protein, and the zinc finger-containing protein Schnurri, are all shown to be required to allow full levels of tinman induction during this process. screw, which encodes a secod BMP2/4-related molecule, has been proposed to act synergistically with dpp to specify dorsal ectoderm and amnioserosa. Similarly, it has been shown that tolloid, which encodes a BMP1-related metalloproteinase, acts to enhance the activity of the dpp gene product during mesoderm induction. Both scw and tolloid are shown to be required for normal induction of tinman expression in the dorsal mesoderm, and in the absence of either gene activity, tinman expression in the dorsal mesoderm is reduced and segmentally interrupted. Thus scw and tld are necessary for achieving full levels of tinman induction, whereas dpp is obligatory for this event. In addition, unlike dpp mutants, mutants for scw or tld form some residual visceral mesoderm. However, heart formation is more sensitive to the activities of scw and tld and is disrupted to a similar extent as in dpp mutants. schnurri is also necessary for tinman induction in the dorsal mesoderm. The dorsal tinman domain is clearly reduced, as compared to wild-type embryos, although the levels of Tinman mRNA are close to normal. Therefore, shn may be required to enhance dpp signaling during tin induction, but significant levels of tin activation can still occur in the absence of its activity (Yin, 1998).

The BMP pathway patterns the dorsal region of the Drosophila embryo. Using an antibody recognizing phosphorylated Mad (pMad), signaling was followed directly. In wild-type embryos, a biphasic activation pattern is observed. At the cellular blastoderm stage, high pMad levels are detected only in the dorsal-most cell rows that give rise to amnioserosa. This accumulation of pMad requires the ligand Screw (Scw), the Short gastrulation (Sog) protein, and cleavage of their complex by Tolloid (Tld). When the inhibitory activity of Sog is removed, Mad phosphorylation is expanded. In spite of the uniform expression of Scw, pMad expansion is restricted to the dorsal domain of the embryo where Dpp is expressed. This demonstrates that Mad phosphorylation requires simultaneous activation by Scw and Dpp. Indeed, the early pMad pattern is abolished when either the Scw receptor Saxophone (Sax), the Dpp receptor Thickveins (Tkv), or Dpp are removed. After germ band extension, a uniform accumulation of pMad is observed in the entire dorsal domain of the embryo, with a sharp border at the junction with the neuroectoderm. From this stage onward, activation by Scw is no longer required, and Dpp suffices to induce high levels of pMad. In these subsequent phases pMad accumulates normally in the presence of ectopic Sog, in contrast to the early phase, indicating that Sog is only capable of blocking activation by Scw and not by Dpp (Dorfman, 2001).

Thus two distinct phases of pMad activation have been identified. The early phase requires activation by both Scw and Dpp ligands, while the second phase depends only on Dpp. Signaling is first detected in the cellular blastoderm embryo. While activation is observed within the dorsal-most 8-10 cell rows, the sensitivity of the detection method fails to monitor signaling in the rest of the dorsal domain. High signaling levels are induced by Scw, and give rise to amnioserosa. Within the domain where pMad is observed, graded activation is detected, which may have the capacity to induce more than one cell fate in the region (Dorfman, 2001).

The cardinal players in the generation of the early pMad gradient are Scw, Tld and Sog. Tld has been suggested to generate a sink for the active ligand, by cleaving the Sog/ligand complex. The similarity between the pMad pattern of scw and tld mutants suggests that Tld is primarily involved in the release of Scw from the complex with Sog. Absence of Scw, Tld or Sax abolished the early pMad pattern while retaining the second phase, indicating that the second phase relies only on Dpp signaling. Similarly, overexpression of Sog eliminated only the early but not the subsequent pMad patterns. This suggests that Sog preferentially associates with Scw, in agreement with previous biological assays of Sog activity. Generation of graded patterning in the dorsal region does not rely on restricted gene expression within this domain. Rather, expression of genes confined to the neuroectoderm may lead to graded distribution of their gene products within the dorsal domain. The essential component for generation of graded patterning appears to be Sog, which is produced only in the neuroectoderm, but is capable of diffusing to the dorsal region. Disruption of the normal distribution of Sog by uniform misexpression, abolishes the early pMad activation profile (Dorfman, 2001).

This suggests that normally Sog may form a graded distribution in the dorsal region, which is essential for patterning. When the Sog/Scw complex is cleaved by Tld, Scw is released and can bind either Sog or Sax. The data suggest that in regions closer to the neuroectoderm, the levels of Sog are high and titrate the free ligand. In the dorsal-most region however, where Sog levels are low, the released Scw has a greater probability of binding and activating the Sax receptor, rather than being trapped again by Sog. Thus, the graded distribution of Sog is critical for generating the reciprocal distribution of Scw, and the ensuing activation profile (Dorfman, 2001).

In sog mutant embryos an expansion of the early pMad pattern is observed. In the absence of Sog, a uniform distribution of Scw is expected, and hence the activation level should be lower than the maximal level in wild-type embryos. The staining levels in wild-type and sog mutant embryos have been quantitated. While the pattern of staining is reproducible in all wild-type embryos, variations in the absolute levels of up to threefold between embryos were observed in any given staining reaction. It is thus difficult to compare reliably the wild-type level to the absolute staining levels of sog mutants. Nevertheless, the impression is that the expanded pMad in sog mutant embryos is comparable in levels to the maximal signaling levels in wild-type embryos. In spite of this expanded pMad activation pattern, amnioserosa cell fates are abolished in sog mutants. This result suggests that in addition to the role of Sog in determining the graded distribution of Scw, Sog or its cleavage products may provide an additional signal facilitating the induction of amnioserosa cell fates (Dorfman, 2001).

Activation of Tkv by Dpp is essential for the appearance of the early pMad pattern, corresponding to the future amnioserosa cells. At this stage, distinct cell fates are also induced in the dorsolateral cells, as reflected by expression of pnr and repression of msh expression. It is assumed that low levels of activation that may be induced by Dpp alone, but not detected by pMad antibodies, are responsible for these fates. Elimination of Dpp or Tkv leads to complete absence of early, as well as late, pMad patterns. Thus, Scw is not sufficient for the early activation phase, and the presence of Dpp is crucial. Cooperativity between Scw and Dpp occurs at the level of receptor activation. One possibility is that the observed pMad levels reflect only an additive effect of Scw and Dpp signaling. Indeed, the number of dpp copies has a profound effect on signaling levels and the shape of the early pMad distribution. Alternatively, it is possible that there is a synergistic interaction between Scw and Dpp signaling. In this case, the requirement of both ligands for the production of the early pMad pattern may indicate that synergy occurs at the level of receptor activation. Phosphorylation of Mad may require the formation of heterotetrameric receptors, containing both Sax/Put and Tkv/Put pairs. Cross linking experiments of the vertebrate receptors support this model (Dorfman, 2001).

Scw is required for generating the pMad pattern only in the early phase. All subsequent patterns rely only on Dpp. This feature may be explained differently by each of the above two models. If Scw and Dpp are required additively in the early phase, higher levels of Dpp may suffice to induce the pMad pattern at later stages. The autoregulatory effects of Dpp on its transcription may account for the elevation in Dpp levels. Alternatively, if Scw and Dpp signaling is synergistic, why is such a synergism necessary only in the early phase? In the early embryo, a maternal transcript encoding an inhibitor of BMP signaling may be translated, to block signaling by Sax/Put or Tkv/Put dimers. Such inhibitor(s) may be displaced only in ligand-bound heterotetrameric receptor complexes. The maternal transcripts of the inhibitor(s) may diminish by stage 9, to allow pMad production by activation of Tkv/Put alone (Dorfman, 2001).

By stage 8/9, Dpp/Tkv activation is sufficient to induce detectable levels of phosphorylated Mad. The second phase of activation does not rely on execution of the early phase, and is detected in scw, tld or sax mutants. A uniform pattern of pMad is observed at this stage within the entire dorsal domain, in accordance with the pattern of autoregulated dpp expression. In the neuroectoderm, brinker (brk) is expressed to suppress Dpp autoregulation. The uniform pMad pattern corresponds to the resulting expression pattern of genes like pannier (pnr) at stage 9, indicating that this second phase of activation is indeed instructive for induction of target genes in the entire dorsal domain. Once cell intercalation leading to germ band extension has been completed, it may be necessary to induce, within the dorsal region, such a uniform activation of Dpp target genes (Dorfman, 2001).

In the second phase, sharp borders of pMad localization are observed, with no detectable activation in the neuroectoderm. Dpp is a diffusible ligand, as indicated by the induction of pMad several cell rows away from the dorsal row of cells expressing Dpp at stage 11. Direct visualization of Dpp in the wing disc has also demonstrated its diffusion capacity over many cell rows. How are the sharp pMad borders generated at stage 9, in view of the diffusability of Dpp? It is suggested that the neuroectoderm cells may produce an inhibitor that prevents activation of the pathway by Dpp molecules that could diffuse from the adjacent dorsal region. Alternatively, the neuroectoderm cells may express cell surface proteins that would block the diffusion of Dpp into the neuroectoderm. When Dpp is expressed ectopically at physiological levels in perpendicular stripes, no pMad activation is observed in the neuroectoderm outside the stripes of Dpp expression. Thus, lower levels of Dpp are not capable of activating the pathway in the neuroectoderm at stage 9 (Dorfman, 2001).

Genetic evidence suggests that the Drosophila ectoderm is patterned by a spatial gradient of bone morphogenetic protein (BMP). Patterns have been compared of two related cellular responses - signal-dependent phosphorylation of the BMP-regulated R-SMAD, MAD, and signal-dependent changes in levels and sub-cellular distribution of the co-SMAD Medea. Nuclear accumulation of Medea requires a BMP signal during blastoderm and gastrula stages. During this period, nuclear co-SMAD responses occur in three distinct patterns. At the end of blastoderm, a broad dorsal domain of weak SMAD response is detected. During early gastrulation, this domain narrows to a thin stripe of strong SMAD response at the dorsal midline. SMAD response levels continue to rise in the dorsal midline region during gastrulation, and flanking plateaus of weak responses are detected in dorsolateral cells. Thus, the thresholds for gene expression responses are implicit in the levels of SMAD responses during gastrulation. Both BMP ligands, DPP and Screw, are required for nuclear co-SMAD responses during these stages. The BMP antagonist Short gastrulation (Sog) is required to elevate peak responses at the dorsal midline as well as to depress responses in dorsolateral cells. The midline SMAD response gradient can form in embryos with reduced dpp gene dosage, but the peak level is reduced. These data support a model in which weak BMP activity during blastoderm defines the boundary between ventral neurogenic ectoderm and dorsal ectoderm. Subsequently, BMP activity creates a step gradient of SMAD responses that patterns the amnioserosa and dorsomedial ectoderm (Sutherland, 2003).

These in vivo studies validate the molecular model for signal-dependent nuclear accumulation of Medea. Nuclear accumulation of Medea requires both competence to oligomerize and MAD. Nuclear accumulation is signal dependent, requiring both BMP ligands, Dpp and Scw. Conversely, all cells accumulated nuclear Medea in the presence of constitutively active Tkv receptor. At these stages, any independent contribution from activin-like signals is below the detection limit (Sutherland, 2003).

Both BMP ligands, Dpp and Scw, are required to form the dorsal-midline gradient. However, scw mutant embryos retain a small amount of dorsal ectoderm, with concomitant expansion of ventral ectoderm. Surprisingly, the weak dorsolateral Medea response is lost in scw embryos. It is concluded that the full Medea response domain encompasses the cell fates that are lost in scw mutants, amnioserosa and dorsomedial ectoderm. It appears that dorsal cells can acquire a dorsolateral fate without gastrula BMP activity (Sutherland, 2003).

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date revised: 12 December 2016

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