How embryonic cells acquire positional information is a key question in developmental biology ., The concept of morphogen gradients , proposed more than a century ago 1 , 2 , has received substantial experimental validation over the past decade ( reviewed in 3 , 4 ) ., Particularly compelling evidence for their existence comes from the identification of secreted proteins that control cell fates in a concentration-dependent manner ., Localized production of Wnt , Hedgehog , and TGF-β family members have been described in numerous tissues and organisms ., However , despite extensive studies on these molecules , the mechanism of transport through tissues and the properties which determine the range of morphogen movement remain poorly understood and controversial ., Here we use the TGF-β family member Decapentaplegic ( Dpp ) in the Drosophila wing imaginal disc as a model to address these issues ., Dpp is expressed in a stripe of anterior compartment ( A ) cells along the anteroposterior ( A-P ) boundary of the wing disc , and forms a concentration gradient along the A-P axis of the wing primordium 5–9 ., Upon binding to the type I-type II/Thick veins ( Tkv ) -Punt receptor complex , the intracellular signal transducer Mothers-against-Dpp ( Mad ) becomes phosphorylated , forms a complex with Medea , and enters the nucleus to inhibit the expression of the transcriptional repressor Brinker ( Brk ) 10–18 ., These events convert the Dpp morphogen gradient into an inverse gradient of Brk activity that mediates many of the patterning and growth functions of Dpp ( 19–21; reviewed in 22 ) ., Although the transduction of the Dpp signal and its role in patterning is well understood , the question of how Dpp is dispersed through its target tissue is still unexplained and thus served as a fertile ground for experimentation and speculations ( reviewed in 23–25 ) ., Several mechanisms for Dpp movement through the wing disc tissue have been proposed ., The simplest model assumes that Dpp disperses by passive extracellular diffusion ., However , because the effective diffusion coefficient of Dpp in the wing disc is three orders of magnitude lower than that of a similarly sized molecule in water 26 , and because a secreted form of GFP fails to form a gradient in wing discs 5 , Dpp gradient formation cannot be explained by free diffusion ., Thus a “restricted extracellular diffusion” ( RED ) model , in which Dpp interacts with its receptor and extracellular matrix ( ECM ) proteins , has been proposed ., This model is supported by theoretical 27 and experimental studies 28 , 29 , which implicate glypicans in the ECM as essential components for Dpp movement ., A completely different mechanism by which Dpp may achieve its long-range distribution is receptor-mediated transcytosis ( RMT ) 5 , 30 ., In this model , Dpp does not move through the extracellular space , but rather through the cell bodies by repeated cycles of endocytosis and re-secretion ., First evidence for this model was gathered from analyzing the Dpp gradient in discs containing shibire mutant cell clones , in which dynamin-dependent endocytosis is blocked ., Entchev et al . ( 2000 ) 5 found reduced Dpp levels “behind” such clones ( i . e . , on the distal side relative to the source ) , suggesting that Dpp is unable to traverse the mutant cells ., Moreover , small lateral clones mutant for tkv also appeared to block Dpp movement 5 , indicating that transcytosis is receptor-mediated ., Although this work has at first been challenged by mathematical modeling and experimental studies 27 , 28 , the transcytosis mechanism was further backed up by theoretical considerations 31 , and by recent work involving FRAP experiments showing that a GFP:Dpp fusion protein is unable to move into a photobleached region when dynamin-dependent endocytosis is blocked 26 ., The two models to explain Dpp movement through an epithelium remain unreconciled , and further analysis is required to determine the contribution of extracellular restricted diffusion or receptor-mediated transcytosis to the formation of the Dpp gradient ., The controversy over Dpp dispersal is augmented by yet another scenario , in which Dpp moves along actin-based filopodia , termed cytonemes , which directly project from the receiving cells to the producing cells 32 , 33 ., Experimental evidence for this mechanism , however , remains elusive , as it is not known yet whether the Dpp ligand is associated with these structures or how a gradient would form along these structures ., Biochemical studies suggest that Dpp binds to the type I receptor Tkv with high affinity 17 , 34 ., Interestingly , all three above mentioned models for Dpp movement rely on the receptor , yet do so in distinct ways ., In the restricted diffusion model , interactions between Dpp and its receptor on the cell surface contribute to the immobilization , subsequent uptake , and degradation of the ligand , thereby impeding Dpp dispersal; in the receptor-mediated transcytosis model the receptor plays an essential role in the uptake ( endocytosis ) and re-secretion ( exocytosis ) of Dpp , and thereby facilitates Dpp movement; and finally in the basic cytoneme model the receptor is used to ferry Dpp along cytonemes ., Here we set out to exploit the pivotal role that the Dpp receptor plays in these mechanisms and manipulated the receptor levels in cell clones to discriminate between the different models of morphogen gradient formation ., We first confirmed in overexpression clones in vivo that Dpp binds to the type I receptor Tkv , but not to the type II receptor Punt ., We then analyzed the effect of tkv mutant clones on the Dpp gradient , and also compared the experimental data to the computed predictions for the RMT and RED models ., While our results challenge the RMT model and are also incompatible with the basic cytoneme mechanism , they are consistent with a RED scenario , in which the majority of Dpp is not bound to Tkv ., Hence we suggest that the major mechanism of Dpp distribution is restricted extracellular diffusion ., The Dpp receptor plays distinct roles for Dpp dispersal in the “restricted extracellular diffusion” ( RED ) and “receptor mediated transcytosis” ( RMT ) models ( see Introduction ) ., Thus , the analysis of Dpp gradient formation in a tissue containing receptor mutant clones promises to discriminate between the two models ., Here we first chose a theoretical approach to investigate the influence of Dpp receptor mutant clones on the Dpp gradient and quantitatively modeled distinct scenarios representing morphogen transport by either the RMT or the RED model ( for a short description of the mathematical modeling , see Box 1; all the analytical details of the model are reported in the Text S1 ) ., Three pools of Dpp ( external-unbound , receptor-bound and internalized ) were described using coupled reaction diffusion equations ., This model has a number of free parameters , which could be constrained though by fixing the relative concentrations of the three Dpp pools , and by the approximation that the Dpp profile exponentially decays outside the production region with a decay length of 20 µm 26 ., We therefore studied limit case scenarios , in which the relative concentrations of the Dpp pools were fixed and Dpp was either mainly internalized ( 80% of total Dpp ) , mainly receptor-bound , or mainly external-unbound ( cf . Box 1 ) ., Our model involves both , pure external diffusion and receptor-mediated transcytosis 35 ., The latter , within its biologically meaningful parameter range ( cf . Box 1 ) , only had an important influence on the total Dpp gradient in the limit case scenario in which Dpp was mainly internalized , and it could be neglected in the other two limit case scenarios ( cf . Box 1 and Text S1 ) ., The RMT model could therefore be represented by the limit case scenario in which Dpp was mainly internalized ( Figure 1A and D ) , and the RED model by the limit case scenarios in which Dpp was mainly receptor-bound ( Figure 1B and E ) or mainly external ( Figure 1C and F ) ., We then modeled the effects of clones containing either a 10-fold increase of receptor levels ( gain-of-function , GOF ) or entirely lacking the receptors ( loss-of-function , LOF ) on the Dpp gradient in the three different scenarios ., The computed Dpp profiles are represented in Figure 1 ., All three transport scenarios predict for the GOF clones an increase of Dpp within the clone territory ., Thus the GOF situations are not suited to discriminate between the RMT and RED models , but they can be exploited to test in vivo which of the Dpp signaling receptors , the type I receptor Tkv or the type II receptor Punt , binds to Dpp ., LOF clones , however , clearly lead to qualitatively different Dpp profiles for each transport scenario ., Most importantly , Dpp levels behind LOF clones are decreased in the RMT model , but are almost unchanged in the RED models ( Figure 1D to 1F ) ., This outcome reflects the necessity of receptors in transporting Dpp by RMT ., Consistent with this , we find that in a scenario in which the majority of Dpp is intracellular , but Dpp is only transported by extracellular diffusion ( the term describing transcytosis is set to zero ) , Dpp levels behind clones are almost unchanged ( Text S1 , Section 5 . 4 ) ., Thus , analyzing Dpp levels behind receptor mutant clones allows us to clearly discriminate between receptor-mediated transcytosis and restricted extracellular diffusion scenarios ., Moreover , analyzing Dpp levels within receptor LOF clones allows one to discriminate between the limit case scenarios in which Dpp is either mainly “receptor-bound” or mainly “external-unbound , ” and quantifying the Dpp levels inside GOF clones allows us to further narrow down the ratio of receptor-bound versus unbound Dpp ., The experimental analysis of the Dpp gradient is complicated by the lack of antibodies that detect the mature , processed form of Dpp ., Visualization , however , can be achieved using a GFP-tagged version of Dpp 5 , 9 ., Because the expression of GFP:Dpp in the dpp expression domain requires the Gal4 system , the generation of receptor GOF clones posed a problem ., We therefore developed LexA-based transgenes that allowed to express GFP:Dpp with a Gal4-independent binary expression system 36 , and employed an actin5c>stop>Gal4 flp-out construct to generate and mark clones overexpressing the Dpp receptor ( Figure 2A ) ., The LexA-based GFP:Dpp gradient resembles the Gal4-based GFP:Dpp gradient ( Figure S1 ) , which has been shown to coincide with the endogenous Dpp activity gradient 5 , 9 , 28 ., The analysis of the receptor LOF clones is hindered by the role of Dpp as a survival and growth factor ., Cells within the wing primordium that lack Dpp signaling activity are efficiently eliminated , in particular when they are located close to the source where Dpp levels are normally high 21 , 37–42 ., This elimination is caused by the upregulation of Brk in Dpp signaling mutant cells ., Hence we sought to prevent this response by genetically generating cells that not only lose Dpp receptor activity but simultaneously also brk function ., However , since the genes encoding Dpp receptors and Brk are located on different chromosome arms , we combined BAC-recombineering and the phiC31 site-specific integration system 43–45 to position a genomic brk rescue construct at chromosomal site 22A , on the same chromosome arm where the type I Dpp receptor tkv is located ., Mitotic recombination at the base of this chromosome arm in a brk mutant background enabled us to generate tkv brk double mutant clones ( for details see Figure 2B ) ., The Dpp ligand signals through the Tkv-Punt typeI-typeII receptor complex ., Upon ligand-receptor binding , Tkv becomes phosphorylated at a glycine/serine rich domain , and in turn phosphorylates and activates Mad 14 , 46 ., While both receptors are necessary for the signal relay , in vitro studies suggest that Dpp binds to Tkv with high affinity , but not to Punt 14 , 17 , 34 ., Here , we reassess these observations in vivo , by analyzing the effect of tkv and punt overexpression on Dpp distribution ., As mentioned before , our theoretical clonal study predicts that any increase in receptor levels will also lead to increased Dpp levels , irrespective of the transport model ( Figure 1A to 1C ) ., To confirm the functionality of the transgenes , we first assessed the levels of Tkv by use of an antibody and estimated that the UAS-tkv transgene results in an approximately 10-fold increase at the protein level ( Figure S2 ) ., We then verified that overexpression of tkv as well as punt ectopically activates Dpp pathway activity by monitoring the phosphorylation state of Mad ( pMad ) ( Figure 3A and 3B ) ., Finally , we analyzed the effect of tkv and put overexpression on the Dpp gradient ., Throughout this work we monitor the Dpp gradient by directly measuring the GFP:Dpp fluorescence intensities ( in green ) and by GFP antibody staining ( in gray ) ., In order to avoid detection of unsecreted Dpp in producing cells and elution of GFP:Dpp from the ECM during fixation , we added the GFP antibody prior to fixation , followed by a 1-hour incubation at room temperature ( for details , see Materials and Methods ) ., Strikingly , only tkv overexpressing clones but not punt overexpressing clones modulate the Dpp profile and lead to an increase of GFP:Dpp levels inside the clones ( Figure 3C and 3D; additional plots for each genotype are shown in Figures S3 and S4 ) ., Thus the comparison of the effect of tkv versus punt overexpression clones on the Dpp profile confirms biochemical studies and suggests that Tkv , but not Punt , binds to Dpp ., Moreover , because the amplification of Dpp signal transduction per se ( also occurred in UAS-punt clones ) does not influence the Dpp profile , we can exclude the possibility that the observed effects in Tkv GOF clones are indirect , and argue that they are a direct consequence of Dpp-Tkv binding ., Although the GOF studies do not enable distinguishing between the RMT and RED models , the different amounts of Dpp in GOF clones can serve to discriminate between the two RED scenarios ., Our data favor the “external-unbound limit case scenario” and suggest that approximately 60%–80% of Dpp is not bound to Tkv ( Figure S4G ) ., As described above , receptor LOF situations were created by simultaneous removal of the receptor and brk ., First we tested whether the alteration of Dpp signaling activity in such clones ( loss of Dpp transduction and loss of brk function ) would affect the Dpp profile , and generated Mad , brk double mutant clones , in which Dpp transduction but not receptor activity is lost ., The Dpp gradient across such clones remains intact ( Figure 4A , B—additional plots are shown in Figure S5 ) ., As a consequence of epithelial folds that occasionally arise at the boundaries of such clones , in some cases a slight modulation of the Dpp profile was observed ( Figure S5 ) ., We then examined the Dpp gradient in discs with tkv , brk LOF clones ., We used the amorphic tkv8 allele , which contains a stop mutation in the extracellular domain of tkv at position 144 of the tkv-PA transcript 47 ., As expected , Dpp signal transduction activity was abolished in tkv− brk− clones ( Figure 5B–C ) ., However , the Dpp gradient in such discs was not significantly altered; tkv− brk− clones resembled Mad− brk− clones ( Figure 4C—additional plots are shown in Figure S6 ) ., The same results were obtained using a conventional antibody staining protocol to detect the Dpp gradient ( Figure S8 ) ., The observation that the Dpp levels inside and behind tkv− clones are not significantly reduced contradicts the receptor-mediated transcytosis model , and concurs with the restricted extracellular diffusion model , in which the majority of Dpp is not bound to Tkv ( see Figure 1 ) ., Apart from Tkv the Drosophila genome encodes another type I receptor , Saxophone ( Sax ) , which has been implicated in Dpp signaling ., Although Sax preferentially interacts with and mediates signaling by the BMP ligand Glass Bottom Boat ( Gbb ) and shows significantly lower affinity to Dpp than Tkv 10 , 34 , 48 , 49 , it could still in principle serve as a Dpp receptor ., To exclude that Sax takes over some functions of Tkv in tkv brk mutant clones , for example shuttling Dpp through mutant cells via receptor-mediated transcytosis , we also analyzed the Dpp gradient in sax null discs containing tkv−brk− clones ( see Materials and Methods ) ., GFP:Dpp levels were not decreased , neither inside nor behind the clones , strengthening our conclusions that GFP:Dpp does not move via receptor-mediated transcytosis ( Figure 4D—additional plots are shown in Figure S7 ) ., Finally we also analyzed the effect of receptor LOF clones on the endogenous Dpp gradient in wing discs ., We tested tkv−brk− and tkv−brk−sax− genotypes and monitored Dpp pathway activity at the level of Mad phosphorylation and target gene expression ., Since Dpp could potentially reach the distal side of such clones by being transported around , rather than through , mutant territory , we purposely collected and analyzed clones with a large dorsoventral extension ., Both readouts , however , show that Dpp signaling is not reduced behind such receptor mutant clones ( Figure 5A–C ) ., To completely eliminate the possibility that this pathway activity stems from Dpp that migrated via clone-surrounding wild-type cells , we identified rare situations where patches of wild-type cells are fully encircled by mutant cells ., As shown in Figure 5C and 5F , even cells in these “islands” exhibit substantial Dpp signaling activity ( for a 3-D reconstruction of these clone islands , see Figure S9 ) ., These findings provide unequivocal evidence that Dpp can disperse through receptor-free territory and hence refute a need for receptor-mediated transcytosis ., Dpp acts as a long-range morphogen , which spreads along the A-P axis of the wing primordium to form a signaling gradient ., Here we studied how receptor mutant clones affect the Dpp gradient in different transport models , and compared theoretical calculations with experimental data ., One outcome of the modeling was the prediction that RMT and RED mechanisms could be discriminated by analyzing Dpp levels behind receptor mutant clones ., While in the transcytosis model these levels should be significantly decreased , they would be almost unaltered in the diffusion model ., This difference stems from the uptake of Dpp by its receptors , which is an essential feature for morphogen transport by RMT , but not by RED ., Our experimental results revealed that neither GFP:Dpp levels nor Dpp signaling activity is reduced behind receptor mutant clones , excluding a significant role for receptor-mediated transcytosis in Dpp gradient formation ., Important support for this conclusion was provided by situations where “islands” of wild-type cells received Dpp signal despite being surrounded by mutant tissue , ruling out the possibility that Dpp reaches the distal side of receptor mutant clones by being transported around the clones ., When analyzing the GFP:Dpp distribution in mosaic tissues , we also found that the Dpp levels are not significantly reduced within receptor mutant clones ., While this outcome further argues against the RMT model , it is consistent with the “external-unbound limit case scenario , ” representing RED with the majority of Dpp not being bound to Tkv ., Indeed , in the GOF experiments the ratio of unbound Dpp could be narrowed down to approximately 60%–80% ., If transcytosis is modeled in a receptor-independent manner ( as shown in Text S1 ) , the effects on Dpp distribution by receptor mutant clones do not differ significantly from those in the restricted extracellular diffusion scenario ., Thus , receptor-independent transcytosis , for example via fluid phase uptake , remains a possible mechanism for Dpp gradient formation ., Several other studies , however , support the restricted extracellular diffusion model ., Based on theoretical grounds , Lander et al . ( 2002 ) 27 proposed that diffusive mechanisms for Dpp gradient formation are more likely than non-diffusive ones ., Moreover , experimental studies on heparan sulfate proteoglycans ( HSPGs ) , in particular glypicans , demonstrated the necessity of an intact ECM for morphogen movement 50 , 51 ., In the Drosophila wing disc , clones mutant for the glypicans Dally and Dally-like ( Dlp ) disrupted the formation of the Dpp gradient 28 ., Dally was also shown to bind Dpp 52 , to stabilize it on the cell surface 53 , and to influence its mobility 54 , 55 ., However , although the evidence that glypicans assist extracellular diffusion of Dpp seems compelling , alternative or additional functions of glypicans in Dpp distribution cannot be excluded ., For example , a recent study 56 suggests that apically localized Dlp binds to the Wingless ( Wg ) morphogen in the Wg producing region , undergoes internalization , and thereby redistributes Wg to the basolateral compartment where Wg spreads to form a long-range gradient ., It is possible that recycling of glypicans is also involved in Dpp relocalization and that this process is important for Dpp movement ., Consistent with such a notion , Kicheva et al . ( 2007 ) 26 reported that dynamin-dependent endocytosis is necessary for Dpp movement ., Blocking such a ubiquitous cellular machinery , however , not only inhibits the recycling of receptors and glypicans , but may also change the composition and distribution of glypicans in the ECM , which in turn might impede extracellular diffusion ., Given that the phenotypes of our receptor clones fully conform to the simplest model of Dpp movement along the ECM ( restricted extracellular diffusion ) , we favor the view that the main function of glypicans for Dpp gradient formation is to facilitate Dpp diffusion along the ECM ., Our observation that receptor mutant clones do not have a major effect on the Dpp gradient contradicts previous observations by Entchev et al . ( 2000 ) 5 ., In their study , ablation of tkv in small lateral clones leads to an accumulation of Dpp at the side of the clone facing the source , arguing for a block of Dpp movement within such clones 5 ., The different results could be explained by the presence of brk in their genetic setup ., The ectopic up-regulation of brk in tkv mutant clones , which in most cases leads to clone elimination 41 , 42 , most likely also causes drastic changes in the transcriptional program in “escaper” cells ., Thus the sharp increase in GFP:Dpp levels at the proximal edge inside tkv mutant clones ( facing the Dpp source ) could be accounted for by increased levels of Dpp binding proteins , a theory which is supported by the fact that Dpp accumulation was strictly clone-autonomous and not in cells ahead of the clones 27 ., In our experimental setup , we avoided such secondary effects by simultaneously removing tkv together with brk ., As our negative control ( Mad brk clones ) shows , the signaling state of these cells ( Dpp signaling off , no Brk ) does not significantly alter the Dpp profile ., Transport along cytonemes is another proposed model for the dispersal of Dpp ( Ramirez-Weber and Kornberg , 1999 ) 33 ., In its simplest implementation , this model assumes that imaginal disc cells form filopodial extensions towards the Dpp producing region and that Dpp is shuttled along these extensions by binding to Tkv 32 ., In this scenario , Tkv GOF clones would not only lead to an increase of receptors inside the clones , but also along the cytonemes , and thus affect the Dpp profile also ahead of the clones ., This , however , was not observed in our experiments ( Figure 3D and Figure S4 ) , and we therefore favor the restricted extracellular diffusion model over the cytoneme model for Dpp gradient formation ., During development morphogens function as short-range or long-range signals in order to specify cell fates within a tissue ., For example , during wing disc development the range of Hh signaling is relatively short compared to that of Dpp , with a functional range of approximately 10 cells versus 40 cells , respectively 7 , 9 , 57 , 58 ., It is likely that properties of the transport system are important determinants of the range of a morphogen ., In the restricted diffusion model , morphogen spreading is impeded by ECM proteins and cell surface receptors , which efficiently trap their ligand at the cell surface and direct it to degradation ., Thus one mechanism to control the range of a morphogen gradient is regulating the receptor levels 27 ., Indeed , the Hh as well as the Dpp system appear to make use of this strategy to regulate their range ., The Hh signal limits its range by upregulating the expression of its binding receptor Patched ( Ptc ) , while the Dpp signal broadens its range by downregulating the expression of its receptor Tkv 6 , 57 , 59 ., The effects of our Tkv LOF and GOF clones on the Dpp profile suggest that the majority of Dpp is not bound to the receptor Tkv ., It is tempting to speculate that the Dpp-Tkv binding properties represent an additional property of the Dpp signaling system that facilitates the formation of a long-range gradient , by assuring that the majority of Dpp remains in a free and unbound state ., Just like lower receptor levels , a lower binding constant would contribute to the spread of Dpp , due to reduced immobilization and degradation of Dpp ., It remains to be seen if the ratio of bound to unbound ligand differs for long- versus short-range morphogens and if this ratio represents a general means to regulate the range of morphogen gradients ., The following transgenes and mutants are described in detail on flybase: UAS-mCherry-CAAX , tub-Gal80ts , hsp70-flp , act5C>y+>Gal4 , arm-lacZ , ubi-GFP ( S65T ) nls , brkM68 , tkv8 , saxP , madB1 , and FRT40 ., Furthermore we used the transgenes: UAS-tkv 59 , UAS-punt 7 , lexO-GFP:Dpp 36 , dpp-LG 36 , dpp-Gal4 5 , and UAS-GFP:Dpp 5 ., In order to introduce the manipulated brk locus into the fly genome , the locus was transferred from the original BAC ( BACR35J16 ) into the attB-Pacman vector , which allows the retrieval of large fragments up to 133 kb ( Venken et al . , 2006 ) 44 ., The BAC clone was ordered from BACPAC Resources , and the BAC DNA isolated according to the protocol provided ., Homology arms of 500 bp , corresponding to the 5′ and 3′ ends of the entire brk genomic locus and spanning parts of the upstream unc-119 gene and the downstream Atg5 gene , were cloned into the attB-Pacman vector ., The attB-Pacman vector was linearized and introduced into recombination-competent SW102 carrying the modified BAC ., The retrieval of the modified DNA fragment into the linearized attB-Pacman was carried out by recombination-mediated gap-repair ., This plasmid was then injected into Drosophila melanogaster embryos ., Site-specific integration of the attB-Pacman vector into the landing site 51D on chromosome 2L was performed as described 43 ., Immunostainings were performed using standard protocols ., Images were collected with a Zeiss LSM710 confocal microscope ., ImageJ was used to analyze the images; z-stacks are shown in maximum projections ., Intensity plots were generated based on the extraction of the intensities in the ROIs and using Mathematica ., For the 3-D reconstruction of z-stacks , Imaris was used .
The TGF-β homolog Decapentaplegic ( Dpp ) acts as a secreted morphogen in the Drosophila wing disc , and spreads through the target tissue in order to form a long range concentration gradient ., Despite extensive studies , the mechanism by which the Dpp gradient is formed remains controversial ., Two opposing mechanisms have been proposed: receptor-mediated transcytosis ( RMT ) and restricted extracellular diffusion ( RED ) ., In these scenarios the receptor for Dpp plays different roles ., In the RMT model it is essential for endocytosis , re-secretion , and thus transport of Dpp , whereas in the RED model it merely modulates Dpp distribution by binding it at the cell surface for internalization and subsequent degradation ., Here we analyzed the effect of receptor mutant clones on the Dpp profile in quantitative mathematical models representing transport by either RMT or RED ., We then , using novel genetic tools , experimentally monitored the actual Dpp gradient in wing discs containing receptor gain-of-function and loss-of-function clones ., Gain-of-function clones reveal that Dpp binds in vivo strongly to the type I receptor Thick veins , but not to the type II receptor Punt ., Importantly , results with the loss-of-function clones then refute the RMT model for Dpp gradient formation , while supporting the RED model in which the majority of Dpp is not bound to Thick veins ., Together our results show that receptor-mediated transcytosis cannot account for Dpp gradient formation , and support restricted extracellular diffusion as the main mechanism for Dpp dispersal ., The properties of this mechanism , in which only a minority of Dpp is receptor-bound , may facilitate long-range distribution .
Morphogens are signaling molecules that trigger specific responses in cells in a concentration-dependent manner ., The formation of morphogen gradients is essential for the patterning of tissues and organs ., Decapentaplegic ( Dpp ) is the Drosophila homolog of the bone morphogenic proteins in vertebrates and forms a morphogen gradient along the anterior-posterior axis of the Drosophila wing imaginal disc , a single-cell layered epithelium ., Dpp determines the growth and final size of the wing disc and serves as an ideal model system to study gradient formation ., Despite extensive studies the mechanism by which morphogen gradients are established remains controversial ., In the case of Dpp two mechanisms have been postulated , namely extracellular diffusion and receptor-mediated transcytosis ., In the first model Dpp is suggested to move by diffusion through the extracellular matrix of a tissue , whereas in the latter model Dpp is transported through the cells by receptor-mediated uptake and re-secretion ., In this work we combined novel genetic tools with mathematical modeling to discriminate between the two models ., Our results suggest that the Dpp gradient forms following the extracellular diffusion mechanism ., Moreover , our data suggest that the majority of the extracellular Dpp is free and not bound to its receptor , a property likely to play a role for the long-range gradient formation .
developmental biology, genetics, biology, computational biology, molecular cell biology, genetics and genomics