magu: Biological Overview | References
Gene name - magu
Synonyms - pent, pentagone
Cytological map position - 46D9-46E1
Function - secreted
Keywords - a secreted protein that acts in a regulatory feedback during establishment and maintenance of BMP/Dpp morphogen signalling during wing development, modifies the ability of cells to trap and transduce BMP by fine-tuning the levels of the BMP reception system at the plasma membrane - internalises the Dpp co-receptors Dally and Dally-like protein - required for Wg signalling, spermatogenesis
Symbol - magu
FlyBase ID: FBgn0262169
Genetic map position - chr2R:10,052,541-10,063,712
Classification - EFh_SPARC_SMOC: EF-hand, extracellular calcium-binding (EC) motif, found in secreted modular calcium-binding protein SMOC-1, SMOC-2, and similar proteins - Thyroglobulin type I repeats - Kazal type serine protease inhibitors and follistatin-like domains
Cellular location - secreted
Tight regulation of signalling activity is crucial for proper tissue patterning and growth. This study investigates the function of Pentagone (Pent/Magu), a secreted protein that acts in a regulatory feedback during establishment and maintenance of BMP/Dpp morphogen signalling during Drosophila wing development. It was shown that Pent internalises the Dpp co-receptors, the glypicans Dally and Dally-like protein (Dlp), and the study proposes that this internalisation is important in the establishment of a long range Dpp gradient. Pent-induced endocytosis and degradation of glypicans requires dynamin- and Rab5, but not clathrin or active BMP signalling. Thus, Pent modifies the ability of cells to trap and transduce BMP by fine-tuning the levels of the BMP reception system at the plasma membrane. In addition, and in accordance with the role of glypicans in multiple signalling pathways, Pent was found to be required for Wg signalling. These data propose a novel mechanism by which morphogen signalling is regulated (Norman, 2016).
Bone morphogenetic protein (BMP) signalling is required in a wide variety of processes across higher organisms, from the establishment of the dorso-ventral (DV) axis in insects to the maintenance of the mammalian gut. Many of the biological functions of BMP signalling require a high degree of spatial regulation; accordingly, multiple mechanisms have evolved that control the movement, stability and activity of BMP ligands (Norman, 2016).
One of the most intensely studied examples of extracellular regulation of BMP comes from Drosophila wing development, a tissue where Dpp (Drosophila BMP2/4) acts as a morphogen to control both patterning and growth. During larval wing development, Dpp is produced in a stripe of cells at the anterior-posterior (AP) boundary and disperses into both compartments, by mechanisms that are still not fully understood, to organize a BMP signalling activity gradient along the AP axis with highest levels in medial and lowest in lateral regions. Dpp, together with a second, uniformly expressed ligand, Glass bottom boat (Gbb), activates membrane bound receptors and induces the phosphorylation of the transcription factor Mad. Phosphorylated Mad (pMad) accumulates in the nucleus with the cofactor Medea, where the activated Smad complex directly regulates BMP-target gene transcription (Norman, 2016).
In addition to the localized production of Dpp, many other determinants impact on proper establishment and maintenance of the activity gradient. Prominent amongst them are membrane-bound BMP-binding proteins, such as Thickveins (Tkv) and Dally, which have dual functions in the establishment of the pMad gradient. Tkv is cell-autonomously required for signalling as it is the main type I BMP receptor in Drosophila. At the same time, Tkv critically affects Dpp tissue distribution through ligand trapping and internalisation, and thus globally shapes the BMP activity gradient. Similarly, Dally, a GPI-anchored heparan sulfate proteoglycan (HSPG), binds and concentrates Dpp at cell surfaces and, together with a second glypican, Dally-like protein (Dlp), is required for both local signal activation and long-range distribution of the ligand. Absence of glypicans- for example in a clone of cells- can result in both a reduction of BMP signalling within the clone and an interruption of Dpp spreading within and beyond the clone. In addition, glypicans can also, by virtue of their ligand-binding capacity, hinder the movement of ligands in several contexts. For example, the diffusion of BMP4 (a vertebrate homolog to Dpp) during early Xenopus embryogenesis has been shown to be restricted through its interactions with HSPGs. Similarly, in the wing disc, increasing the levels of Dally at the source of Dpp causes a local increase of signalling activity and a drastic compaction of the gradient due to ligand trapping. Reflecting the importance of the activity of Tkv and Dally for proper gradient establishment, the levels of both proteins are tightly regulated along the AP axis of the developing disc. Through complex transcriptional regulation, which involves repression by BMP signalling itself, both Tkv and Dally are down-regulated near the ligand source to maintain the proper balance between Dpp signalling and Dpp dispersion (Norman, 2016).
Pentagone (Pent; also known as Magu), is an additional determinant in the establishment, maintenance and scaling of the BMP signalling gradient in the developing wing (Ben-Zvi, 2011; Hamaratoglu, 2011; Li, 2009; Vuilleumier, 2010; Zheng, 2011). The transcription of pent is directly repressed by BMP signalling, hence its production is restricted to the lateral-most cells of the disc. Pent protein is, however, secreted and distributes in a gradient that is inverse to the pMad gradient. Pent mutants have a restricted pMad gradient with abnormally high levels in the centre of the disc and very low levels in lateral regions; consequently, adult wings have growth and patterning defects in lateral regions. The pMad gradient of pent mutants thus resembles the abnormal gradients caused by medial over-expression of Tkv or Dally, suggesting an interaction of the protein with the BMP-reception system. Indeed, past work established that Pent physically associates with Dally on cell membranes, but the consequences of this interaction have remained unclear (Norman, 2016).
This study presents data showing that Pent binds and induces the internalisation of both Drosophila glypicans, resulting in reduction of Dally and Dlp protein levels. Endocytosis of glypicans is dependent on dynamin and Rab5, but does not require clathrin or Dpp signalling. Additionally, it was shown that Pent influences Wg signalling, which also depends on glypicans. It is concluded from these data that Pent modulates glypican levels in order to modify multiple signalling pathways during wing morphogenesis. The data suggest an additional, protein-level feedback mechanism to tightly control levels of signalling, which cooperates with transcriptional regulatory feedback loops to ensure proper morphogen gradient formation and organ development (Norman, 2016). It has been proposed that the function of Pent in Dpp gradient formation could be to either enhance the ability of Dally to displace Dpp, or to reduce the co-receptor function of Dally (Vuilleumier, 2010). From the data presented in this study, it is proposed that Pent reduces the co-receptor function of glypicans by binding them and inducing their internalisation. It is suggested that Pent may promote spreading of Dpp by reducing Dpp co-receptors and therefore Dpp trapping and signal transduction. Internalisation of glypicans is independent of signalling and Dpp itself, which fits the model that removal of glypicans by Pent enhances spreading. Furthermore, this study has presented data showing that by regulating glypican levels, Pent is also able to modulate Wingless signalling (Norman, 2016).
The work proposes that Pent modulates Dpp signalling via the co-receptors Dally and Dlp. The relative contribution of Dally and Dlp to Dpp signalling is unknown, although both must be removed in order for a reduction in pMad to occur. Data showing that Pent influences the co-receptor but not the receptor itself distinguishes Pent from other BMP signalling modifiers in D. melanogaster, such as Crossveinless-2, Short gastrulation and Twisted gastrulation, which bind either the BMP ligand, the receptor, or both. This could reflect the different roles that BMP signalling must fulfill in D. melanogaster, where it forms a long-range gradient in the larval wing disc but short-range gradients in the embryo and pupal wing (Norman, 2016).
The data show that Pent binds and internalises glypicans. Prior to endocytosis, it is probable that glypicans are clustered at the cell surface by Pent, and this might also inhibit their function without necessarily inducing their internalisation. Glypicans share physical properties, notably a GPI anchor and heparan sulphate side chains, upon which Pent binds. This study has shown that internalisation of glypicans by Pent requires dynamin and Rab5 but not clathrin. Cell culture experiments have shown that GPI proteins are commonly endocytosed via clathrin independent mechanisms, but this has not been demonstrated before in Drosophila. Many clathrin independent mechanisms have been described in cultured cells, but in vivo evidence for many of them is lacking. Lipid-rich microdomains, in some cases marked by flotillin, can be involved, but this study found no requirement for flotillin in the endocytosis of glypicans by Pent (Norman, 2016).
One of the key problems cells must overcome to internalise GPI anchored proteins is that they have no cytoplasmic region to mediate recruitment into endocytic pits. A similar process to that described in this study, the Hh mediated internalisation of GPC3, is thought to utilise LRP1 in order to communicate with the endocytic machinery. This does not seem to be the case with Pent and Dally, as knockdown of the Drosophila homologue of LRP1 does not affect internalisation. It is possible that protein clustering, and the membrane deformations this has been predicted to cause, may be involved in the internalisation of glypicans by Pent. The precise mechanism by which Pent internalises glypicans will be an interesting avenue of future research (Norman, 2016).
While the data implicate glypicans as the direct target of Pent's activity, it cannot be ruled out that the effect on Dpp gradient formation involves the regulation of Tkv levels and/or activity. While Pent does not bind to Tkv directly and Pent over-expression seems not to affect the levels of membrane bound Tkv, a substantial amount of the receptor is found in Pent- and Dally positive endocytic vesicles. This raises the possibility that Pent might target a specific subpopulation of Tkv for degradation, and that this interaction requires glypicans as adaptors (Norman, 2016).
The data show that Pent binds and internalises Dally and Dlp. Glypicans, in particular Dally, have been shown to regulate the spreading of Dpp, in addition to being essential for Dpp signal transduction itself. The molecular basis for these activities is the binding of Dpp to the heparan sulphate side-chains of glypicans, probably a first step that serves to concentrate Dpp at the surface of the disc epithelium. Glypican-bound Dpp molecules can follow multiple routes, as they can be passed to receptors (promoting signalling), to glypicans of neighbouring cells (promoting ligand dispersion), or can persist on glypicans of the same cell resulting in local ligand enrichment. It is probable that the specific outcome at any position along the morphogen field will depend on the relative levels and activities of the involved factors, i.e. the ligand, receptors and glypicans. A similar balance between glypicans, receptors and ligands has been proposed to explain the biphasic activity of Dlp in Wg signalling in the wing imaginal disc. In the case of Dpp, levels of glypicans need to be tightly regulated to allow for the optimal balance between ligand release, trapping and receptor binding. The data suggest that Pent contributes to this balance by fine-tuning the levels of glypicans. It is proposed that in the absence of Pent, glypican levels are too high and this results in excessive ligand trapping and enhanced local signalling. Such local effects would be accompanied with a non-autonomous reduction in ligand spreading and shrinkage of the pMad gradient. An approximation mimicking this situation is artificially elevating levels of Dally in medial regions, which has been shown to locally increase pMad. This study has shown that this increase in signalling by Dally is at the expense of Dpp spreading to the rest of the disc and the formation of the long range pMad gradient. This clearly shows that excess Dally can block spreading of Dpp. Notably, pent mutants display a similarly compacted activity gradient with high medial and low lateral pMad levels. Importantly, ligand-binding properties of HSPGs have been described to impede ligand spreading in multiple physiological contexts, including BMP4 in Xenopus early dorso-ventral patterning and FGF10 in its role in branching morphogenesis (Norman, 2016).
Multiple transcriptional feedback loops are required for the maintenance of the Dpp signalling gradient in the wing. Primary amongst these is the repression of Tkv and Dally transcription by Dpp signalling. This ensures that receptor and co-receptor levels are low near the Dpp producing cells, allowing Dpp to spread out from the centre of the disc. These feedback loops are important for proper establishment of the Dpp signalling gradient. However, such direct feedbacks targeting the production of molecules with ligand-binding properties may have limitations. In response to a reduction in spreading of Dpp, Tkv and Dally levels would increase to locally compensate the reduction in Dpp signalling activity. Such an increase would, however, further enhance trapping and internalisation of the ligand and, at the level of the whole wing disc, would further block Dpp spreading. From the data it is suggested that Pent, a secreted negative regulator of Dpp signalling, fine-tunes the signalling gradient at a different level, by directly adjusting glypican levels and reducing the inbuilt increase in co-receptor and ligand-trapping upon a reduction in the extent of the pMad gradient. This might happen at a critical region of the wing disc, the mediolateral cells, where declining levels of the spreading ligand face increasing levels of the receptor and co-receptor (Tkv and Dally, respectively). Pent, secreted by lateral cells next to this region, could reduce the glypican pool to allow Dpp to overcome excessive ligand trapping and thus promote further spreading. Consistent with such a 'remote' activity, Pent can be detected throughout the wing disc. As Pent is transcriptionally repressed by Dpp signalling and, unlike Tkv and Dally, does not bind Dpp, Pent might be a good candidate for how the system overcomes the inherent limitations of feedback loops involving membrane tethered, Dpp-binding proteins (Norman, 2016).
The key extracellular signalling molecules of the wing disc, Dpp, Wg and Hh, all bind to glypicans. The regulatory proteins Pent, Notum and Shifted also bind glypicans, putting glypicans at the centre of signalling regulation in the wing disc. Consequently, any factor that affects glypican function, such as Pent, is likely to modify multiple signalling pathways. This study has shown that Pent is also able to influence Wg signalling, thus providing a possible link between the Wg and Dpp pathways (Norman, 2016).
The role of glypicans in Wg signalling is well described and complex. Dlp can stimulate Wg signalling, Wg accumulates on cells over-expressing Dlp and fails to accumulate on cells mutant for Dlp. Similarly, the data show that excess Pent internalises glypicans and reduces extracellular Wg. Precise in vitro assays have shown that low levels of Dlp enhance Wg signalling, but too much Dlp reduces signalling. Furthermore, recent evidence shows that deacylation of Wg by Notum, which reduces Wg signalling activity, requires glypicans. It is clear, then, that the level of glypicans must be very finely balanced for Wg signalling to be at the correct level. It is proposed that the elevated glypican levels observed in the absence of Pent push this fine balance towards inhibition of signalling, due to the increased levels of glypicans sequestering Wg away from the receptor and also increasing the platform upon which Notum can deacylate Wg. Consistent with this conclusion, the effects of Notum and Dlp over-expression can be suppressed by increasing the level of Pent protein (Norman, 2016).
Interestingly, inactivation of the BMP-response elements in the regulatory region of the pent gene locus results in prominent expression of pent at the DV boundary (Vuilleumier, 2010), hinting at an input into pent transcription from DV signals. Future studies, including quantitative studies and modelling, should give further insight into pathway interaction and coordination during tissue development by molecules such as Pent (Norman, 2016).
A model is proposed that Pent internalises glypicans to modify multiple signalling pathways. Future work should address the influence of Pent on glypican organisation at the nanoscale, and also the type of membranes at which Tkv and Dally localise, questions that are challenging to answer using current methods. In order to fully understand the role of Pent in establishment of the long range Dpp gradient, first it is important to gain a better understand how glypicans function in Dpp signalling and how Dpp is spread throughout the tissue (Norman, 2016).
The wing of the fruit fly, Drosophila melanogaster, with its simple, two-dimensional structure, is a model organ well suited for a systems biology approach. The wing arises from an epithelial sac referred to as the wing imaginal disc, which undergoes a phase of massive growth and concomitant patterning during larval stages. The Decapentaplegic (Dpp) morphogen plays a central role in wing formation with its ability to co-coordinately regulate patterning and growth. This study asked whether the Dpp signaling activity scales, i.e., expands proportionally, with the growing wing imaginal disc. Using new methods for spatial and temporal quantification of Dpp activity and its scaling properties, it was found that the Dpp response scales with the size of the growing tissue. Notably, scaling is not perfect at all positions in the field and the scaling of target gene domains is ensured specifically where they define vein positions. It was also found that the target gene domains are not defined at constant concentration thresholds of the downstream Dpp activity gradients P-Mad and Brinker. Most interestingly, Pentagone (Pent/Magu), an important secreted feedback regulator of the pathway, plays a central role in scaling and acts as an expander of the Dpp gradient during disc growth (Hararatoglu, 2011).
This study measured Dpp pathway activity using an antibody specific to the phosphorylated form of Mad, and compared the P-Mad levels in space and time with the activity levels of direct target genes, such as brk, which plays key roles in both growth and patterning. Transcription of brk is directly repressed via P-Mad binding at defined silencer elements (SEs), resulting in inversely graded brk expression. Brk is the only known regulator affecting the positioning of the expression boundary of omb, while sal and dad translate input from both P-Mad and Brk into their expression boundaries. The dynamics of all of these readouts were analysed using antibodies where possible, to avoid potential misinterpretations due to reporter stability (Hararatoglu, 2011).
P-Mad levels scale very well posterior to 0.4 Lp (Lp stands for the length of the posterior compartment) with the exception of TC5 profiles near the D/V boundary. Previous studies that examined P-Mad scaling reached contradictory conclusions: the P-Mad gradients in late stage discs were reported to correlate with tissue size in a previous study and to have no correlation in another. Similarly, examination of P-Mad gradients across discs of different sizes led to the conclusion that the gradient did not expand, but in another study the P-Mad gradient was shown to scale with tissue size. It is believed that most of the confusion can be attributed to different profile extraction protocols as well as to the use of various definitions of scaling, as discussed below (Hararatoglu, 2011).
Since P-Mad is an early signature of the activation of the Dpp signaling pathway, it was of interest to find out how its scaling properties translate to its immediate key target, the brk gene. In addition to brk being directly repressed by P-Mad, the Brk protein itself is necessary for graded brk transcription. The range of Brk expression was found to strictly follow P-Mad in both wild-type and pent mutant discs. Similar to what was observe for P-Mad, Brk also shows very good scaling for positions posterior to 0.4 Lp. By contrast, levels of Brk increase steadily as the discs grow and cannot be explained by P-Mad dynamics alone. This increase in Brk levels could be due to the build-up of the unknown activator of brk transcription or, alternatively, the SEs in brk could become desensitized to the repressive input of P-Mad. Regardless of the cause of the increase, cells at a given relative position experience increasing levels of the Brk repressor over time (Hararatoglu, 2011).
Traditionally, Dpp and P-Mad gradients have been described by a decaying exponential with characteristic decay length λ. This decay length is different for each profile and corresponds to the position at which the protein levels have decreased by a factor e. The correlation between the decay length and the tissue size has been used as a proxy for scaling, e.g. one study found no significant scaling for the Dpp and P-Mad profiles at the end of 3rd instar stage. Similarly, the width of the P-Mad profile has been used to characterize the spread of the gradient and it was concluded that the width of the P-Mad profile is constant during growth. In contrast, the current study detected that the P-Mad profile expands as the tissue grows. This discrepancy may be due to the fact the previous study lacked time classes TC3 and TC4 in their sample collection, the period where the P-Mad gradient expands before contracting again when measured close to the D/V boundary. Thus, possibly the measurements of the P-Mad profiles were done in the vicinity of the D/V boundary, where at TC5 P-Mad has a sharp profile reminiscent of 30 h younger discs. Hence, the choice of position can significantly alter the final interpretations of the data (Hararatoglu, 2011).
Consistent with these results, another study (Wartlick, 2011) recently showed that the decay lengths of Dpp-GFP, P-Mad, brk-GFP, and dad-RFP do correlate with the length and the area of the posterior compartment during tissue growth. Importantly, the Wartlick study also assessed scaling qualitatively in the whole field by looking at the collapse of the profiles in relative positions and normalized intensities. This method has two advantages: it does not require any fitting of the profiles, and it shows scaling at all positions and not just at the characteristic decay length position (i.e., x = λ) (Hararatoglu, 2011).
A recent mathematical model termed 'expansion-repression integral feedback control' suggests that scaling can emerge as a natural consequence of a feedback loop (BenZvi, 2010). The hypothetical 'expander' molecule facilitates the spread of the morphogen and in turn is repressed by it; scaling is achieved given that the expander is stable and diffusible. The known properties of Pent fit the requirements of this hypothetical agent: Pent is secreted, required for Dpp spreading, and pent transcription is directly inhibited by Dpp signaling. However, it is not known how stable Pent is, and pent transcription is never abolished in the entire field in which the gradient acts during larval stages. To test whether Pent could be a key player involved in scaling of Dpp activity during disc growth, the analyses were repeated at all time points in the absence of Pent. It was found that the P-Mad and Brk gradients indeed fail to scale with the tissue size in this mutant background. Scaling of dad-GFP and Omb are also strongly affected, while Sal still exhibits some degree of Pent-independent scaling in the anterior compartment. Importantly, while the function of Pent is essential for proper scaling of the Dpp activity gradient, it is noted that Pent alone cannot account for the observed selective scaling of Omb and Sal domain boundaries. Scaling of these target genes specifically in those regions in which they have a patterning function points to the involvement of additional players, which will be the subject of future research. Hence, the current findings strongly suggest that Pent is a very good candidate to be the expander in the 'expansion-repression integral feedback control' model and therefore provide the first mechanistic insights into the question of scaling in wing patterning. The exact biochemical functions of Pent have to be determined in order to get a more mechanical view of gradient scaling in the developing wing imaginal disc (Hararatoglu, 2011).
More than 40 years ago, Lewis Wolpert proposed the French flag model to explain pattern formation by morphogens. This study tested whether the activity gradients downstream of Dpp, namely P-Mad and Brk, are read out by their target genes at constant concentration thresholds. Thus, average P-Mad and Brk concentrations were measured at Dad, Omb, and Sal expression boundaries across development. It was found that the amount of P-Mad at these boundaries slightly increased (Dad), slightly decreased (Sal), or was constant throughout development (Omb). Among these three targets, the Omb domain is the widest and it corresponds to a region where the P-Mad gradients scale perfectly; as a result, P-Mad levels fluctuate very little at the Omb domain boundary. Interestingly, the domain boundary of Omb is thought to solely depend on Brk and hence constant P-Mad levels might be a mere coincidence. Remarkably, all the target genes that were considered respond to significantly increasing levels of Brk, suggesting that the target genes desensitize to Brk over time, so that more and more Brk can be tolerated at the domain boundary. Alternatively, if it is considered that the domain boundaries of dad-GFP and Sal do not respond to constant P-Mad levels either, another explanation could be that Brk and P-Mad signals are combined in a non-additive fashion in order to define the boundary position of the target genes. Following this assumption, a simple combination of these signals was sought that is constant at the target gene domain boundary for all TCs. It is proposed that Brk and the unknown activator of Omb could be similarly combined in order to determine the Omb domain boundary (Hararatoglu, 2011).
This paper's data was used to further test a model that was recently proposed to explain the uniform growth in the wing imaginal disc (Wartlick, 2011). The model poses that the temporal changes in Dpp signaling levels drive tissue growth; cells divide when they experience a relative increase of 50% in the levels of Dpp signaling. Since it is the relative differences and not the absolute amount of Dpp signal that regulate cell divisions, the model can account for the uniform growth of the wing disc. Since the relative increase in Dpp activity slows down, the cell cycles lengthen as the disc grows. Growth stops when the cell division time exceeds 30 h. The model of Wartlick is based on the finding that Dpp activity scales with tissue size and that cells at a given relative position experience increasing levels of Dpp signaling over time. In contrast, a general temporal increase was not observed in the level of Dpp signaling at a given relative position in this study. P-Mad is the most upstream and the most dynamic readout available for the activity of the Dpp pathway and it was found that the relative increase in P-Mad levels throughout development is not significantly different from zero at most relative positions. Why is the increase in Dpp-GFP levels not reflected in P-Mad levels? A potential explanation for this might be that the observed accumulation of Dpp-GFP was due to the stability and accumulation of Gal4 since Dpp-GFP was under UAS control. The Wartlick study showed that the half-life of the Dpp-GFP fusion protein is only 20 min, but the Gal4 stability was not considered. Alternatively, the system could get desensitized over time and more and more Dpp would be required to lead to similar P-Mad levels. Finally, increases in Dad levels could counteract the increase in Dpp levels, since Dad is an inhibitory Smad (Hararatoglu, 2011).
Wartlick (2011) monitored Dpp signaling levels using a dad-RFP reporter and found a 5-fold increase in the course of 36 h. In the current analysis, a similar tool, dad-GFP, was used and the Wartlick results could not be fully reproduced. Though it was also found that dad-GFP scales with tissue size, its levels increase merely 2-fold over 40 h, and this increase takes place only in the medial 25% region of the disc, while cells in the lateral part experience a decrease in dad-GFP levels. This disparity in the fold increases is likely due to the higher stability of RFP, since the enhancer used was identical in both studies. Additionally, it was found that the levels of Brk, another direct target of the pathway with a very well-established role in suppressing growth in lateral regions, increase in average 4-fold in the interval studied, an observation not reported by Wartlick. In the lateral areas, increase in Dpp activity (if present) is below detection levels and would be opposed by increasing Brk levels. Importantly, increasing Brk levels, if they were to depend solely on Dpp, would suggest decreasing Dpp activity in lateral areas as Brk expression is directly suppressed by Dpp activity. Hence, the data raise serious questions about the validity of this uniform growth model, especially in the lateral regions of the pouch. An alternative model is favored that does not rely on Dpp activity alone to explain uniform growth in the wing disc (Hararatoglu, 2011).
Understanding how stem cells are maintained in their microenvironment (the niche) is vital for their application in regenerative medicine. Studies of Drosophila male germline stem cells (GSCs) have served as a paradigm in niche-stem cell biology. It is known that the BMP and JAK-STAT pathways are necessary for the maintenance of GSCs in the testis. However, recent work strongly suggests that BMP signaling is the primary pathway leading to GSC self-renewal (Leatherman, 2010). This study shows that magu controls GSC maintenance by modulating the BMP pathway. magu, a putative matricellular protein, a secreted protein that could regulate cell-matrix interactions, is specifically expressed from hub cells, and accumulates at the testis tip. Testes from magu mutants exhibit a reduced number of GSCs, yet maintain a normal population of somatic stem cells and hub cells. Additionally, BMP pathway activity is reduced, whereas JAK-STAT activation is retained in mutant testes. Finally, GSC loss caused by the magu mutation is suppressed by overactivating the BMP pathway in the germline (Zheng, 2011).
This study shows that magu plays an important role in GSC maintenance. Strong evidence is provided that it does so by modulating BMP activation in germ cells. magu encodes a secreted protein of the SPARC/BM-40/osteonectin family, recently shown to ensure the proper activity gradient for the BMP morphogen, Dpp, across the developing wing epithelium (Vuilleumier, 2010). The role characterized for magu in the testis niche exhibits some similarities as well as differences to that proposed for the wing (Zheng, 2011).
It has been shown that the BMP pathway is activated and required in GSCs, whereas the JAK-STAT pathway is activated and required in both GSCs and CySCs. The data shows that magu is required for maintenance of GSCs, but not CySCs, and that BMP activation was impaired in germ cells adjacent to the hub in magu mutants. It was also found that forcing activation of the BMP pathway in germ cells substantively rescues the magu phenotype. Thus, it is concluded that the primary role of magu in the testis niche is to modulate BMP signaling and thereby maintain GSCs (Zheng, 2011).
Superficially, these results suggest that magu works in a manner similar to that described in the wing epithelium, where magu facilitates the transport of BMP ligands to establish the proper signaling gradient. However, there are several differences comparing the wing with the testis niche (Zheng, 2011).
The most obvious is that to control wing patterning, BMP signaling is graded and must be effective over a long range. Thus, Dpp is expressed from a stripe of cells in the center of the wing disk, while the region where BMP activation is modulated by magu is located far laterally, many tens of cells away from the ligand source (Vuilleumier, 2010). In striking contrast to this situation, BMP ligands are produced in hub cells and CySCs of testes, which are directly adjacent to GSCs, where pathway activation is required. In the testis, there is no documented graded requirement, and, if anything, it is likely that pathway activation must be restricted to cells near the niche to ensure that few cells take on stem cell character. Therefore, while magu is thought to assist the movement of Dpp over a long range in the wing (Vuilleumier, 2010), there is no need for long-range transport for GSC maintenance in the testis. This distinction between the two systems suggests that key mechanistic differences remain to be uncovered for how magu affects BMP signaling (Zheng, 2011).
One way that magu supports robust signaling far from the BMP ligand source in the wing is that magu gene expression is engaged by a feedback circuit in order to be used as a positive modulator of signaling. Thus, magu expression is repressed in areas of relatively high signaling, and that repression is relieved in regions of low signaling. Its action in the low signaling region is to promote signaling even though these areas are far from the ligand source (Vuilleumier, 2010). In fact, expressing magu ectopically in the area of high signaling serves to dampen signaling there, while enhances signal at a distance, presumably by promoting movement or stabilization of the ligand. In the testis niche, there is some evidence for feedback regulation, as a reporter construct mutated for pMad/Medea/Schnurri complex binding sites (Vuilleumier, 2010) is expressed more robustly, and in more hub cells. However, in contrast to the wing, there is no evidence that this negative feedback regulation is necessary in the testis niche, as overexpression of magu had no discernable effect on GSC numbers (data not shown) (Zheng, 2011).
One other potential difference between the wing and testis niche is that the BMP ligands acted on by magu might differ in the two systems. Vuilleumier haS addressed the function of magu with respect to Dpp, the principal BMP ligand used globally for wing patterning. However, the major BMP ligand for male GSC maintenance appears to be Gbb. This difference could have consequences for the mechanism by which magu influences BMP signaling comparing the two systems. For example, although Dpp does not interact directly with magu (Vuilleumier, 2010), the potential remains that magu might bind to Gbb for GSC maintenance (Zheng, 2011).
In this regard, it is worth noting that gbb is expressed throughout the wing, and that compromising gbb function does generate a wing vein phenotype similar to magu mutants. Thus, in the wing, even though the focus has been on Dpp, perhaps there is an effect also on Gbb transport and/or signaling. Thus, further investigation of the modulation of BMP signaling by magu in both the wing and testis niche should be revealing (Zheng, 2011).
The fact that overexpressing a constitutively active form of BMP type I receptor in the germline can rescue the GSC phenotype suggests that magu acts upstream of receptor binding. This is in agreement with its proposed role in the wing and also preliminary analysis in zebrafish (Vuilleumier, 2010). There are a number of membrane-associated and secreted factors that magu might influence to modulate BMP signaling (Zheng, 2011).
In the wing, magu interacts directly with Dally, a HSPG (heparan sulfate proteoglycan) (Vuilleumier, 2010). Interestingly, Dally and its homologue Dally-like (Dlp) are also important for male GSC maintenance. While no genetic interactions has been found between magu and dally, dlp or several other genes needed for HSPG biosynthesis, some preliminary data indicate that overexpressing dlp in the germ cells can increase the fraction of testes retaining GSCs among magu mutants. Dlp has been shown to be enriched among hub cells, this study has had no success in reproducing this suggestive distribution. Therefore, further experiments are needed to test for interactions between magu and Dlp or other HSPGs in GSC maintenance (Zheng, 2011).
Given that magu is secreted from hub cells, its localization could have suggested a more specific hypothesis for its action in the testis niche. However, magu protein localization among cells of the niche appears complex. An antibody raised against an N-terminal portion of magu exhibits punctate signal restricted among hub cells, and at the hub-GSC interface, but this serum was effective only sporadically. A second serum directed against a C-terminal peptide (Vuilleumier, 2010) robustly exhibits the same punctate pattern among hub cells, but also reveals a slightly extended distribution among stem cells and their daughters near the hub. Additionally, this serum revealed strong punctate signal likely among the extracellular matrix (ECM) near the hub. It is not possible at this time to distinguish whether the pool of magu associated with ECM or the more generally distributed pool is active for GSC maintenance (Zheng, 2011).
However, considering the close proximity of hub cells to GSCs, it is simplest to envision that magu acts along the hub cell-germline stem cell interface, where the interaction of BMP ligands and receptors occurs. It is possible that magu facilitates interactions between BMPs and their receptors via formation of ternary ligand/magu/receptor complexes. This model has been shown for Crossveinless 2 (Cv2), an extracellular BMP modulator engaged for crossvein patterning in the wing. Cv2 can also bind to Dally, and the Cv2-HSPG interaction is likely important for normal BMP signaling in crossvein patterning. magu and its vertebrate orthologues SMOC1/2 have two Thyroglobulin type-1 repeats. It has been shown that proteins with such repeats can inhibit extracellular proteases. Thus, although Cv2 appears to have no effect on the function of Tolkin, the protease promoting BMP signaling in crossvein patterning, it is reasonable to speculate that magu may function as a protease inhibitor to protect BMP ligands from being degraded by extracellular proteases (Zheng, 2011).
Alternatively, the enrichment observed among the ECM is interesting. Among the family of proteins to which magu belongs, SPARC interacts with type IV Collagen, a component of basement membranes, and SMOC1/2 are associated with basement membranes. Interestingly, Viking (Vkg), the type IV collagen in Drosophila, is involved in the female GSC maintenance, potentially by sequestration of Dpp, thereby restricting BMP signaling in the germarium. It would be interesting to investigate whether Vkg also plays a similar role in the testis, and interacts with magu to maintain a normal number of GSCs (Zheng, 2011).
Maintaining a proportionate body plan requires the adjustment or scaling of organ pattern with organ size. Scaling is a general property of developmental systems, yet little is known about its underlying molecular mechanisms. Using theoretical modeling, this study examined how the Dpp activation gradient in the Drosophila wing imaginal disc scales with disc size. It is predicted that scaling is achieved through an expansion-repression mechanism whose mediator is the widely diffusible protein Pentagone (Pent). Central to this mechanism is the repression of pent expression by Dpp signaling, which provides an effective size measurement, and the Pent-dependent expansion of the Dpp gradient, which adjusts the gradient with tissue size. This mechanism was validated experimentally by demonstrating that scaling requires Pent and further, that scaling is abolished when pent is ubiquitously expressed. The expansion-repression circuit can be readily implemented by a variety of molecular interactions, suggesting its general utilization for scaling morphogen gradients during development (Ben-Zvi, 2011).
In many instances during development, morphogens specify cell fates by forming concentration gradients. In the Drosophila melanogaster wing imaginal disc, Decapentaplegic (Dpp), a bone morphogenetic protein (BMP), functions as a long-range morphogen to control patterning and growth. Dpp is secreted from a stripe of cells at the anterior-posterior compartment boundary and spreads into both compartments to generate a characteristic BMP activity gradient. Ever since the identification of the morphogen activity of Dpp in the developing wing, the system has served as a paradigm to understand how long-range gradients are established and how cells respond to such gradients. This study reveals the tight and direct connection of these two processes with the identification and characterization of pentagone (pent), a transcriptional target of BMP signalling encoding a secreted regulator of the pathway. Absence of pent in the wing disc causes a severe contraction of the BMP activity gradient resulting in patterning and growth defects. Pent was shown to interact with the glypican Dally to control Dpp distribution and provide evidence that proper establishment of the BMP morphogen gradient requires the inbuilt feedback loop embodied by Pent (Vuilleumier, 2010).
During Drosophila aging mortality rate increases exponentially and progeny production per animal declines dramatically, correlating with decreased number and division of somatic and germ-line stem cells in the gonads. To search for genes that might promote both longevity and fecundity, a P element transposon (PdL), containing an outwardly directed, doxycycline-inducible promoter was used to generate conditional mutations. Mutant females were screened for increased fecundity at late ages in the presence of doxycycline. Two genes were identified, named hebe (CG1623) and magu (CG2264), that when over-expressed in adult flies could increase life span by approximately 5%-30% in both sexes and increase female fecundity at late ages. Transcripts for magu are enriched in the Drosophila stem cell niche region, and magu encodes a protein related to the human SMOC2 regulator of angiogenesis. While moderate over-expression of magu in adult females increased fecundity at late ages, high-level over-expression of magu was maternal-effect lethal. The data demonstrate that adult-specific over-expression of hebe and magu can increase life span and modulate female fecundity, and provide further evidence against obligatory trade-offs between reproduction and longevity (Li, 2009).
The matricellular protein SMOC (Secreted Modular Calcium binding protein) is conserved phylogenetically from vertebrates to arthropods. It has been previously shown that SMOC inhibits bone morphogenetic protein (BMP) signaling downstream of its receptor via activation of mitogen-activated protein kinase (MAPK) signaling. In contrast, the most prominent effect of the Drosophila orthologue, pentagone (pent), is expanding the range of BMP signaling during wing patterning. Using SMOC deletion constructs this study found that SMOC-∆EC, lacking the extracellular calcium binding (EC) domain, inhibits BMP2 signaling, whereas SMOC-EC (EC domain only) enhances BMP2 signaling. The SMOC-EC domain binds HSPGs with a similar affinity to BMP2 and can expand the range of BMP signaling in an in vitro assay by competition for HSPG-binding. Together with data from studies in vivo the study proposes a model to explain how these two activities contribute to the function of Pent in Drosophila wing development and SMOC in mammalian joint formation (Thomas, 2017).
Search PubMed for articles about Drosophila Magu or Pentagone
Ben-Zvi, D., Pyrowolakis, G., Barkai, N. and Shilo, B. Z. (2011). Expansion-repression mechanism for scaling the Dpp activation gradient in Drosophila wing imaginal discs. Curr Biol 21(16): 1391-1396. PubMed ID: 21835621
Hamaratoglu, F., et al. (2011). Dpp signaling activity requires Pentagone to scale with tissue size in the growing Drosophila wing imaginal disc. PLoS Biol. 9(10): e1001182. PubMed Citation: 22039350
Leatherman, J. L. and DiNardo, S. (2010). Germline self-renewal requires cyst stem cells and stat regulates niche adhesion in Drosophila testes. Nat. Cell Biol. 12: 806-811. PubMed Citation: 20622868
Li, Y. and Tower, J. (2009). Adult-specific over-expression of the Drosophila genes magu and hebe increases life span and modulates late-age female fecundity. Mol Genet Genomics 281(2): 147-162. PubMed ID: 19011900
Norman, M., Vuilleumier, R., Springhorn, A., Gawlik, J. and Pyrowolakis, G. (2016). Pentagone internalises glypicans to fine-tune multiple signalling pathways. eLife 5:e13301. PubMed ID: 27269283
Thomas, J. T., Eric Dollins, D., Andrykovich, K. R., Chu, T., Stultz, B. G., Hursh, D. A. and Moos, M. (2017). SMOC can act as both an antagonist and an expander of BMP signaling. eLife 6:e17935. PubMed ID: 28323621
Vuilleumier, R. (2010). Control of Dpp morphogen signalling by a secreted feedback regulator. Nat. Cell Biol. 12: 611-617. PubMed Citation: 20453847
Wartlick, O., et al. (2011). Dynamics of Dpp signaling and proliferation control. Science 331: 1154-1159. PubMed Citation: 21385708
Zheng, Q., Wang, Y., Vargas, E. and DiNardo, S. (2011). magu is required for germline stem cell self-renewal through BMP signaling in the Drosophila testis. Dev. Biol. 357(1): 202-10. PubMed Citation: 21723859
date revised: 2 August 2018
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