ࡱ> 555`5a5b5c5d5e5f5g5h5i5j5k5l5m5n5o5p5q5r5s5t5u5v5w5x5y5z5{5|5}5~5555555555555555555555555555555555555555555555555555555555555555555555555555555q`  @bjbjqPqP (j::%~~~Z8Z8Z888 9\?6:v|="===arӐ,$uh9~Oa==eQBj=L~=Rt z~T=9 -iZ8h0?ini<TTpi~vTD%R^?!1D1 Project Progress Summary: Aim 1a: What Controls the Formation of Morphogen Gradients? During the initial phase of embryonic development, identical cells simply divide to reproduce more of the same. At some stage, signaling protein molecules known as morphogens (aka ligands) are synthesized at a localized site. These morphogen molecules are transported from their production site; some bind to their cell receptors along the way, generally resulting in different receptor occupancies at different cell locations. The spatial concentration gradient of morphogen-receptor complexes (aka bound morphogens) induces spatially graded differences in cell signaling. The differential cell signaling in turn gives rise to different gene expressions from which follows different stable cell fates and visually patterned arrangements of tissues and organs. Although the strategy of using bound morphogen gradients [LR] to create pattern is conceptually simple (as demonstrated in  ADDIN EN.CITE Lander20021180Lander, A. ,Nie,Q. , Wan, F.2002Do morphogen gradients arise by diffusionDevelopment Cell26785-796(Lander 2002) for the BMP family member Dpp in the wing imaginal disc of Drosophilas), the need to build gradients of appropriate sizes, shapes and rates of formation imposes some constraints. For example, the tendency of signaling receptors to degrade their ligand (of concentration [L]) can create a need for morphogen receptors to have slow association kinetics, and to be expressed at low levels. In fact, there was considerable uncertainty about diffusion as a mechanism for transporting ligand away from the (localized) production site (see  ADDIN EN.CITE Lander20021180Lander, A. ,Nie,Q. , Wan, F.2002Do morphogen gradients arise by diffusionDevelopment Cell26785-796(Lander 2002) and references therein). To the extent that morphogen gradients must be biologically capable of inducing different cell signaling at different cell locations and thereby different cell fate, limitations are imposed on the range of acceptable system parameter values such as ligand and receptor synthesis rates, VL and VR, and binding, dissociation, and degradation rate constants, kon, koff, and kdeg, as shown in Figure 1 (with  = VL/(D/Xmax2) being a scaled ligand synthesis rate and  = konRtot/(D/Xmax2)/(1+ koff/kdeg) a scaled effective binding rate).  SHAPE \* MERGEFORMAT  Figure 1 Some of the constraints on system parameters are caused by the simplifying approximation of the biological processes being modeled. For the Dpp gradient in the wing disc investigated in  ADDIN EN.CITE Lander20021180Lander, A. ,Nie,Q. , Wan, F.2002Do morphogen gradients arise by diffusionDevelopment Cell26785-796(Lander 2002), these include modeling the ligand synthesis by a point source while in reality it extends over several cell widths and ignoring the flux across the edge of the ligand production region. (One surprising result of our early effort was that the boundary value problem for determining the steady state gradient in an extracellular model, without ligand-receptor internalization and with or without receptor renewal, has the same mathematical form as that with endocytosis (see  ADDIN EN.CITE Lou20042620Lou, Y. , Nie, Q., Wan, F.2004Nonlinear Eigenvalue Problems in the Stability Analysis of Morphogen GradientsStudies in Applied Mathematics113183-215(Lou 2004)). Hence, if we are only interested in steady state gradients, we can work with the extracellular model by properly interpreting the system rate constants (see also  ADDIN EN.CITE Lander20052670Lander, A. , Nie, Q. , Wan, F.2005cInternalization and End Flux in Morphogen Gradient FormationAccepted, J. of Computational and Applied Math.Available online 6 June, 2005(Lander 2005c). Model Refinements (Lander, Nie, Vargas and Wan): In the early research activities supported by the grant being renewed, the point source idealization was removed and the consequences of a distributed Dpp production region of a few cell width around the compartment boundary of the wing disc were studied in  ADDIN EN.CITE Lander2005a2690Lander, A. D., Nie, Q. , Wan, F.Y.M.2005aSpatially Distributed Morphogen Production and Morphogen Gradient FormationMathematical Biosciences and Engineering2239-262(Lander 2005a). The principal effect of a distributed source is the assured existence of a steady state signaling gradient for the entire range of morphogen synthesis rate. To relate this spatially distributed source model to a model with a point source, we aggregated the distributed source and showed that for a relatively narrow ligand production region (compared to the wing disc span) the distributed source model may be adequately approximated by a point source model  ADDIN EN.CITE Lander20052680Lander, A. , Nie, Q. , Vargas, B. Wan, F.2005bAggregation of Distributed Sources in Morphogen Gradients FormationStudies in Applied Mathematics1144343-374(Lander 2005b). The boundary condition at the source end so derived corresponds to the one used in  ADDIN EN.CITE Lander20021180Lander, A. ,Nie,Q. , Wan, F.2002Do morphogen gradients arise by diffusionDevelopment Cell26785-796(Lander 2002) (see also  ADDIN EN.CITE Lou20042620Lou, Y. , Nie, Q., Wan, F.2004Nonlinear Eigenvalue Problems in the Stability Analysis of Morphogen GradientsStudies in Applied Mathematics113183-215(Lou 2004)) except for the neglected flux term in the earlier effort. The aggregated source model was analyzed in  ADDIN EN.CITE Lander20052680Lander, A. , Nie, Q. , Vargas, B. Wan, F.2005bAggregation of Distributed Sources in Morphogen Gradients FormationStudies in Applied Mathematics1144343-374(Lander 2005b) with the results delimiting the range of applicability of those from the earlier efforts without the end flux term  ADDIN EN.CITE Lander20021180Lander, A. ,Nie,Q. , Wan, F.2002Do morphogen gradients arise by diffusionDevelopment Cell26785-796(Lander 2002), ADDIN EN.CITE Lou20042620Lou, Y. , Nie, Q., Wan, F.2004Nonlinear Eigenvalue Problems in the Stability Analysis of Morphogen GradientsStudies in Applied Mathematics113183-215(Lou 2004). The results reported in  ADDIN EN.CITE Lander20052680Lander, A. , Nie, Q. , Vargas, B. Wan, F.2005bAggregation of Distributed Sources in Morphogen Gradients FormationStudies in Applied Mathematics1144343-374(Lander 2005b) assure us that the unexpected phenomenon of stable locally intense ligand-receptor concentration gradients observed in some recent experiments is theoretical possible and can be described by relatively simple mathematical relations. At high ligand synthesis rates, the signaling gradients are generally not appropriate for multi-fate cell developments; they are too flat over a large portion of the signaling gradient region and hence not multi-fate gradients. At moderate ligand synthesis rate, receptors will cause gradients to decay too rapidly with distance if receptor level is not low or rate of binding to receptors is not slow. To expand the region of the parameter space for multi-fate gradients, more complex models of gradient formation were then developed to include additional ligand activities (such as the effects of non-signaling molecules that bind with the relevant ligand, feedback regulations, and co-reception) not previously considered. We report here mainly on the progress made on the effects of non-receptor sites (and leave other effects to preliminary results sections below). Modeling Non-Receptor Binding Sites: Morphogens can bind much more than just cell surface receptors. For example, on vertebrate cells, BMP-2 and 4 (the orthologs of Dpp) bind to low affinity sites that are at least as, if not more abundant than receptor sites. Abundant low affinity sites are also seen with other morphogens, (e.g. Wnts and FGFs [56-57]). The low affinity of such sites is often quite substantial. For BMPs (as well as Wnts and FGRs), many such non-receptor sites are accounted for by cell surface heparan sulfate proteoglycans (HSPGs). In the Drosophila wing disc, HSPGs are not only present, they affect Dpp signaling  ADDIN EN.CITE Jackson1997130Jackson, S.M.Nakato, H.Sugiura, M.Jannuzi, A.Oakes, R.Kaluza, V.Golden, C.Selleck, S.B1997<styles><style face='2'></style><style start='27'></style><style face='2' start='32'></style><style start='42'></style></styles>division abnormally delayed, a Drosophila glypican, controls cellular responses to the TGF-ß-related morphogen, Dpp.Development1244113-4120(Jackson, Nakato et al. 1997). For the morphogen hedgehog (hh), HSPGs have been specifically implicated in morphogen transport (although the mechanisms underlying this effect are unknown  ADDIN EN.CITE The1999530The, I.Bellaiche, Y.Perrimon, N.Hedgehog movement is regulated through tout velu-dependent synthesis of a heparan sulfate proteoglycanAnimalBiological MarkersDrosophila/embryologyFibroblast Growth Factor/metabolismGene Expression RegulationHeparan Sulfate Proteoglycan/*biosynthesis/metabolismImmunohistochemistryInsect Proteins/*metabolismMembrane Proteins/*genetics/metabolismProto-Oncogene Proteins/metabolismSignal TransductionSupport, Non-U.S. Gov'tMol Cell199944633-9.(The, Bellaiche et al. 1999)). With the current grant support, we have examined how non-receptor binding sites affect the establishment of morphogen gradients in several ways. Results from two of our projects are reported under this Aim; others will appear as preliminary results in later sections: a) Addition of Cell Surface Bound Non-receptors to Existing Dpp Wing Disc Models (Lander, Nie & Wan): We can modify the models B, R and C in  ADDIN EN.CITE Lander20021180Lander, A. ,Nie,Q. , Wan, F.2002Do morphogen gradients arise by diffusionDevelopment Cell26785-796(Lander 2002),  ADDIN EN.CITE Lou20042620Lou, Y. , Nie, Q., Wan, F.2004Nonlinear Eigenvalue Problems in the Stability Analysis of Morphogen GradientsStudies in Applied Mathematics113183-215(Lou 2004) to include an additional set of association, dissociation, and internalization/degradation reactions associated with a cell surface bound non-receptor (such as HSPG members). For example, modifying model B (with a fixed receptor concentration Rtot and non-receptor concentration Ntot) yields: (A/(t = D'(2A/(x2 konRtotA(1 B) + koffB jonNtotA(1 zC) + joffC (B/(t = konRtotA(1 B) (koff + kdeg)B (1) (C/(t = jonNtotA(1 zC) - (joffB + jdeg)C with z = Rtot/Ntot and jon, joff and jdeg being association, dissociation and internalization/ degradation rate constants (respectively) associated with morphogen binding to non-receptor sites. In Eq. (1), A=[L]/Rtot and B = [LR]/Rtot represent concentrations of free and receptor-bound morphogen whereas C = [LN]/Rtot represents the concentration of morphogen bound to non-receptor sites. Similar modifications can be made to the equations in models R and C (see Preliminary Results section). The modified system B, designated as model NB has been analyzed in  ADDIN EN.CITE Lander20052770Lander, A.D., Nie, Q. and Wan, F.Y.M.2005dMembrane associated non-recptors and morphogen gradients, Bulletin of Math.Bulletin of Mathematical BiologyAccepted for Publication (http://www.math.uci.edu/~fwan/pdf/BulletinMB.pdf)(Lander 2005d). Among the results reported therein, we showed the effects of the addition of non-receptor sites help to explain why overexpression of morphogen receptors can have different (and sometimes opposite) effect in different morphogen systems. Known experimental results show that when Dpp receptors thick veins (tkv) in wing imaginal discs are overexpressed in vivo  ADDIN EN.CITE Lecuit1998270Lecuit, T.Cohen, S. M.Dpp receptor levels contribute to shaping the Dpp morphogen gradient in the Drosophila wing imaginal discAnimalDNA Probes/geneticsDrosophila/*embryologyFluorescent Dyes/metabolismGene Expression Regulation, Developmental/geneticsIn Situ HybridizationInsect Proteins/*metabolismMicroscopy, FluorescenceMorphogenesis/*physiologyProtein-Serine-Threonine Kinases/*geneticsReceptors, Cell Surface/*geneticsSignal TransductionWing/*growth & developmenthttp://www.ncbi.nlm.nih.gov/cgi-bin/Entrez/referer?http://www.biologists.com/Development/125/24/dev2353.html http://www.biologists.com/Development/125/24/dev2353.htmlDevelopment1998125244901-7.(Lecuit and Cohen 1998)], they produce effects significantly different from (and nearly opposite to) those of overexpressing the Wingless (Wg) receptor Drosophila frizzled 2 (Dfz2)  ADDIN EN.CITE Cadigan19982780Cadigan, K.M., Fish, M.P., Rulifson, E.J., and Nusse, R.1998Wingless repression of Drosoplila frizzled 2 expression shapes the Wingless morphogen gradient in the wingCell93767-777(Cadigan 1998). More specifically, we have from  ADDIN EN.CITE Lecuit1998270Lecuit, T.Cohen, S. M.Dpp receptor levels contribute to shaping the Dpp morphogen gradient in the Drosophila wing imaginal discAnimalDNA Probes/geneticsDrosophila/*embryologyFluorescent Dyes/metabolismGene Expression Regulation, Developmental/geneticsIn Situ HybridizationInsect Proteins/*metabolismMicroscopy, FluorescenceMorphogenesis/*physiologyProtein-Serine-Threonine Kinases/*geneticsReceptors, Cell Surface/*geneticsSignal TransductionWing/*growth & developmenthttp://www.ncbi.nlm.nih.gov/cgi-bin/Entrez/referer?http://www.biologists.com/Development/125/24/dev2353.html http://www.biologists.com/Development/125/24/dev2353.htmlDevelopment1998125244901-7.(Lecuit and Cohen 1998) that "high levels of tkv generally sequester ligand and limit its movement across the wing disc." The corresponding ligand-receptor gradient becomes steeper and more convex, tending to a boundary layer, while the amplitude remains pretty much unchanged. On the other hand, we have from  ADDIN EN.CITE Cadigan19982780Cadigan, K.M., Fish, M.P., Rulifson, E.J., and Nusse, R.1998Wingless repression of Drosoplila frizzled 2 expression shapes the Wingless morphogen gradient in the wingCell93767-777(Cadigan 1998) that "high levels of a Wg receptor, Drosophila frizzled 2 (Dfz2), stabilizeWg, allowing it to reach cells far from its site of synthesis." The theoretical results from model NB  ADDIN EN.CITE Lander20052770Lander, A.D., Nie, Q. and Wan, F.Y.M.2005dMembrane associated non-recptors and morphogen gradients, Bulletin of Math.Bulletin of Mathematical BiologyAccepted for Publication (http://www.math.uci.edu/~fwan/pdf/BulletinMB.pdf)(Lander 2005d) show that at a high concentration of non-receptor to that of receptor such as that of Wg receptor Dfz2 in  ADDIN EN.CITE Cadigan19982780Cadigan, K.M., Fish, M.P., Rulifson, E.J., and Nusse, R.1998Wingless repression of Drosoplila frizzled 2 expression shapes the Wingless morphogen gradient in the wingCell93767-777(Cadigan 1998), overexpression of signal receptor does not change the spread of the signaling gradient, only the amplitude of concentration at the source edge But for thick veins with konRtot/(D/Xmax2) << jonNtot/(D/Xmax2), the model predicts that overexpression of the receptor would result in steeper and more convex [LR] gradient as observed experimentally in  ADDIN EN.CITE Lecuit1998270Lecuit, T.Cohen, S. M.Dpp receptor levels contribute to shaping the Dpp morphogen gradient in the Drosophila wing imaginal discAnimalDNA Probes/geneticsDrosophila/*embryologyFluorescent Dyes/metabolismGene Expression Regulation, Developmental/geneticsIn Situ HybridizationInsect Proteins/*metabolismMicroscopy, FluorescenceMorphogenesis/*physiologyProtein-Serine-Threonine Kinases/*geneticsReceptors, Cell Surface/*geneticsSignal TransductionWing/*growth & developmenthttp://www.ncbi.nlm.nih.gov/cgi-bin/Entrez/referer?http://www.biologists.com/Development/125/24/dev2353.html http://www.biologists.com/Development/125/24/dev2353.htmlDevelopment1998125244901-7.(Lecuit and Cohen 1998). Thus, although intuition might suggest that non-receptor binding sites, by competing with receptors, can only inhibit the formation of gradients, the results of our investigation showed that if the rate constants associated with binding, dissociation, internalization and degradation are very different for non-receptor and receptor binding sites, then a variety of behaviors is possible as we had anticipated in our original grant proposal. More theoretical and numerical prediction by models incorporating effects of non-receptor (bound or unbound to a cell surface) will be reported under other Aims below and in the Preliminary Result section. b) Effects of heparan sulfate proteoglycan Syndecan on Wingless Signaling (Marcu, Marsh, Nguyen, Nie & Wan) We turn next to our theoretical and experimental investigation of how the non-receptor Syndecan promotes internalization of Wingless and inhibits wingless signaling by overexpressing Syndecan. To mimic mathematically the overexpression of syndecan, we varied the rate and region of syndecan production within the time limit of 30 hours chosen to see an effect on the wingless gradient. When Vs was increased 3 times (5x10-5 to 5x10-2) the formation of WR2 (Wingless-Frizzled2) signaling complexes decreased 6.25 times (0.25 to 0.04). This is consistent with the biological observation that overproduction of syndecan, in an area covering the production region of wingless, leads to loss of signaling. To determine the time evolution of gradients, we note that the experimental overproduction of syndecan (high Vs) has the observed effect of transiently expanding the dorso-ventral gradient of wg. This is explained by the increase in wg production (increased Vw) following the relief of negative feedback of wg on its own production. Mathematically however, the evolution of the relevant gradients from time 0-30 hours following the increase in Vs shows that the gradient narrows rather than expanding. This would be expected from the overproduction of a proteoglycan which does not have an intracellular component, like the glypicans dally and dally-like. If such a proteoglycan is to be produced in the region of wingless expression, the surface sugar chains would trap wingless outside the cell and prevent diffusion, thus sharpening the gradient. In fact we do observe this effect of sharpening the gradient when we overexpress a truncated form of syndecan which lacks the cytoplasmic tail (Figure (2)). Since our model for the full-length syndecan does not yet include the internalization of wg through the syndecan cytoplasmic domain, the result fits our prediction. With internalization followed by degradation we expect a longer time to reach steady-state and a transient expansion of the gradient followed by reduction of the signal. To show mathematically and biologically the effect of loss of syndecan (with no genetic mutant which allows studying the wingless gradient), we proposed 2 approaches: (i) using the mathematical model with Vs = 0 and thus mimic a null mutant, and (ii) look at the loss of syndecan in flies which express siRNA for syndecan. The second had been done for the different isoforms of syndecan. The result confirmed the prediction that in the absence of syndecan there will be a gain of wingless signaling, as shown (in Figure (3)) by the 85-100% rescue of wing margin notches in the animals in which siRNA knocks off syndecan function (B, female expressing syndecan siRNA) vs. controls (A, male lacking the transgene). To uncover the molecular mechanism of syndecan activity We proposed that the antagonistic function of Syndecan is due to its function as either a nonreceptor for Wingless or as a co-receptor with R3, and suggested that this function is possibly mediated by PDZ proteins which act as linkers between Syndecan and the Frizzled receptors. To date we have produced and purified bacterial extracts containing the individual and tandem PDZ domains of these proteins and monitored their interaction with the Syndecan and Frizzled cytoplasmic tails. The data obtained so far shows that at least one protein binds Fz2 through one of its PDZ domains (Figure 4). So far, we have considered only the non-receptor model mathematically as suggested by the interaction with R2. We are currently testing the co-receptor model with a set of equations that includes internalization. Experimentally we will do competition assays in the presence of Sdc and Frizzled to determine whether the interaction is cooperative or exclusive. Figure (2) The wg gradient sharpens in the presence of syndecan lacking the cytoplasmic tail  SHAPE \* MERGEFORMAT  Figure (3) Loss of syndecan produces gain of wingless signaling  A B Figure (4) GM6509 PDZ2 binds Fz2 (R2) Dilutions of the purified protein are shown next to the curve. Binding is measured in Response Units (RU) over time after washing out the extract run over the flow cell.  SHAPE \* MERGEFORMAT  A manuscript on the experimental aspects of the problem has been written but is awaiting for the completion the theoretical study before submission for publication (see  ADDIN EN.CITE Marcu20052830Marcu, O., Agrawal, N. , Heslip, T. and Marsh, J.L.2005 Syndecan modulates the distribution and signaling of Wingless morphogen in the Drosophila wing marginPreprint (http://www.math.uci.edu/~fwan/sdc-wg.doc/html)(Marcu 2005),  HYPERLINK "http://www.math.uci.edu/~fwan/sdc-wg.doc/html" http://www.math.uci.edu/~fwan/sdc-wg.doc/html) Extension to Higher Dimension: Morphogen transport in fields such as fly wing discs occurs in an essentially two-dimensional space. Because the Dpp source in the disc is a linear array of cells in the center of the disc, and we are primarily interested in the formation of gradients perpendicular to this line (e.g., in the case of Dpp, along the antero-posterior axis), we have been treating morphogen gradient formation as a one-dimensional problem in our initial modeling efforts, e.g., System NB in Eq. (1). It was proposed as a part of Aim 1 that we would extend our analysis to explicitly include the second dimension to see how the additional geometrical feature would affect the theoretical conclusions and predictions of the 1-D models. As expected, results from our two-dimensional treatment (e.g.,  ADDIN EN.CITE Lander200315320Lander, A. Nie, Q. , Wan, F., Xu, J.2003Diffusion and Morphogen Gradient FormationSubmitted (http://math.uci.edu/~fwan/pdf/all.pdf)66 pages(Lander 2003)) show that the 1-D models are found to capture the essential features of the system, including the approach to steady state (see Figure (5)) away from the edge of the wing disc (or its idealized boundaries). We have also results for other geometries, including morphogen movement on the surface of a spherical shell, modeling the frog or fly embryo, being prepared for publication.  Figure (5) Aim 1b: How do inhibitors of Dpp signaling such as Sog and Tsg increase Dpp activity at the embryonic dorsal midline?(Lander, Marsh, Nie, Wan and Zhang) The phenotypes of Twisted gastrulation (Tsg) and Short gastrulation (Sog) mutants indicate that they are required to maintain a high level of Dpp signaling at the dorsal midline of the embryo (Figure (6)). Reconciling this fact with observations that Tsg and Sog together act as potent inhibitors of Dpp signaling is not straightforward. Recent reviews offer intuitive explanations invoking effects of Tsg and Sog on Dpp transport  ADDIN EN.CITE Ross2001660http://www.ncbi.nlm.nih.gov/htbin-post/Entrez/query?db=m&form=6&dopt=r&uid=11260716Ross, J. J.Shimmi, O.Vilmos, P.Petryk, A.Kim, H.Gaudenz, K.Hermanson, S.Ekker, S. C.O'Connor, M. B.Marsh, J. L.Twisted gastrulation is a conserved extracellular BMP antagonist[1] Departments of Genetics, Cell Biology and Development, [2] Howard Hughes Medical Institute, [3] These authors contributed equally to this work.11260716Nature20014106827479-83.Harland2001590http://www.ncbi.nlm.nih.gov/htbin-post/Entrez/query?db=m&form=6&dopt=r&uid=11260695Harland, R. M.Developmental biology A twist on embryonic signalling11260695Nature20014106827423-4.Ray2001650http://www.ncbi.nlm.nih.gov/htbin-post/Entrez/query?db=m&form=6&dopt=r&uid=11290316Ray, R. P.Wharton, K. A.Twisted Perspective. New Insights into Extracellular Modulation of BMP Signaling during DevelopmentDepartment of Molecular Biology, Cell Biology and Biochemistry, Division of Biology and Medicine, Box G-J160, Brown University, 02912, Providence, RI, USA11290316Cell20011046801-804.(Harland 2001; Ray and Wharton 2001; Ross, Shimmi et al. 2001), Our modeling and simulation studies  ADDIN EN.CITE Mizutani20052300Mizutani, C.,Nie,Q.,Wan, F.,Zhang, Y. ,Vilmos, P. ,Bier, E., Marsh,L., and Lander, A.2005Formation of the BMP activity gradient in the Drosophila embryoDevelopmental Cell8June-61 - 10(Mizutani 2005) have yet to provide support for such a mechanism. It has been proposed that Tsg+Sog bind and carry Dpp to a dorsal location where it is released--potentially by the action of the secreted protease Tolloid (Tld)--thereby concentrating Dpp in the dorsal midline (and lowering its level laterally  ADDIN EN.CITE Ross2001660http://www.ncbi.nlm.nih.gov/htbin-post/Entrez/query?db=m&form=6&dopt=r&uid=11260716Ross, J. J.Shimmi, O.Vilmos, P.Petryk, A.Kim, H.Gaudenz, K.Hermanson, S.Ekker, S. C.O'Connor, M. B.Marsh, J. L.Twisted gastrulation is a conserved extracellular BMP antagonist[1] Departments of Genetics, Cell Biology and Development, [2] Howard Hughes Medical Institute, [3] These authors contributed equally to this work.11260716Nature20014106827479-83.Harland2001590http://www.ncbi.nlm.nih.gov/htbin-post/Entrez/query?db=m&form=6&dopt=r&uid=11260695Harland, R. M.Developmental biology A twist on embryonic signalling11260695Nature20014106827423-4.Ray2001650http://www.ncbi.nlm.nih.gov/htbin-post/Entrez/query?db=m&form=6&dopt=r&uid=11290316Ray, R. P.Wharton, K. A.Twisted Perspective. New Insights into Extracellular Modulation of BMP Signaling during DevelopmentDepartment of Molecular Biology, Cell Biology and Biochemistry, Division of Biology and Medicine, Box G-J160, Brown University, 02912, Providence, RI, USA11290316Cell20011046801-804.(Harland 2001; Ray and Wharton 2001; Ross, Shimmi et al. 2001)). We undertook a research project to test this hypothesis and examine the conditions need to be met for a concentrating effect by modeling the diffusion, binding and turnover of Dpp, Sog, Tolloid (Tld) and Tsg in the fly embryo by Eq. (2) (Figure (7)), designated as System STT, in much that same way as we did for Dpp in the wing disc. With this description we have obtained both analytical results and numerical simulations (see  ADDIN EN.CITE Lou20051550Lou, Y. , Nie, Q., Wan, F.2005Effects of Sog on Dpp-Receptor BindingSIAM J. Appl. Math. (http://math.uci.edu/~fwan/download/LNW-SIAM.pdf)6551748 - 1771(Lou 2005), ADDIN EN.CITE Mizutani20052300Mizutani, C.,Nie,Q.,Wan, F.,Zhang, Y. ,Vilmos, P. ,Bier, E., Marsh,L., and Lander, A.2005Formation of the BMP activity gradient in the Drosophila embryoDevelopmental Cell8June-61 - 10(Mizutani 2005)) on how various system parameters affect distribution of the Dpp ligand and its occupied receptors. With gradient formation and action occurring over a very short time period in the Drosophila embryo, the biologically important solutions to this system will be at times well before the establishment of any steady state. Hence, an understanding on how the gradients evolve is significant to our quest for insight to mechanism underlying the observed phenomena.    (2)  In  ADDIN EN.CITE Mizutani20052300Mizutani, C.,Nie,Q.,Wan, F.,Zhang, Y. ,Vilmos, P. ,Bier, E., Marsh,L., and Lander, A.2005Formation of the BMP activity gradient in the Drosophila embryoDevelopmental Cell8June-61 - 10(Mizutani 2005), we used experimental and computational approaches to investigate how the interactions of a multi-protein network create the unusual shape and dynamics of formation of this gradient. Starting with the observation suggesting that receptor-mediated BMP degradation is an important driving force in gradient dynamics, we developed a general model that is capable of capturing both subtle aspects of gradient behavior and a level of robustness that agrees with in vivo results. The results reported in  ADDIN EN.CITE Lou20051550Lou, Y. , Nie, Q., Wan, F.2005Effects of Sog on Dpp-Receptor BindingSIAM J. Appl. Math. (http://math.uci.edu/~fwan/download/LNW-SIAM.pdf)6551748 - 1771(Lou 2005), ADDIN EN.CITE Mizutani20052300Mizutani, C.,Nie,Q.,Wan, F.,Zhang, Y. ,Vilmos, P. ,Bier, E., Marsh,L., and Lander, A.2005Formation of the BMP activity gradient in the Drosophila embryoDevelopmental Cell8June-61 - 10(Mizutani 2005) assure us that the unexpected phenomenon of stable locally intense ligand-receptor concentration gradients observed in recent experiments is theoretical possible and can be described by relatively simple mathematical relations. Aim 2: To investigate the effects of the role of inhibition/activation and feedback on cell signaling (Lander, Nie and Wan) With the current grant support, we investigated, both theoretically (by mathematical modeling, analysis and numerical simulations) and experimentally, how do feedback interactions enable morphogen gradients to specify complex, stable and robust patterns. The considerable amount of results obtained will be reported under Preliminary Results of Aim 1 of the new proposal. We mention here only that a large part of these results have been reported in a manuscript submitted for publication  ADDIN EN.CITE Lander20052810Lander, A.D., Wan, F.Y.M., Heather M. Elledge, H.M., Claudia Mieko Mizutani, C.M., Bier, E. and Nie, Q.2005eDiverse Paths to Morphogen Gradient RobustnessSubmitted for publication (http://www.math.uci.edu/~fwan/pdf/DivPaths.pdf)(Lander 2005e). Others, such as the possibility of a positive feedback destabilizing the normally steady gradients by lengthening the decaying time of transients, will be reported in manuscripts under preparation (including one cited under Aim 1a  ADDIN EN.CITE Marcu20052830Marcu, O., Agrawal, N. , Heslip, T. and Marsh, J.L.2005 Syndecan modulates the distribution and signaling of Wingless morphogen in the Drosophila wing marginPreprint (http://www.math.uci.edu/~fwan/sdc-wg.doc/html)(Marcu 2005)). Aim 3: To test the predictions emerging from aims 1-2 abov. In addition to experimental work reported under the previous Aims, a team headed by Co-PI Marsh performed experiments on diffusive rate for Dpp in Drosophila wing discs to establish Fluorescence Correlation Spectroscopy measurements in tissues. The results are published  ADDIN EN.CITE Wang20042290Wang, Z., Marcu, O., Berns, M.W. and Marsh, J. L.2004In vivo FCS measurements of ligand diffusion in intact tissueJ. of Biomedical Optics9(2)(Wang 2004). Fluorescence correlation spectroscopy (FCS) is a powerful tool for studying the dynamic properties of diffusion and chemical reactions, as well as molecular concentrations.1 Recent developments in FCS technology have opened the possibility to observe the in vivo properties of protein molecules2-6. To date, in vivo FCS studies are mainly performed on single living cells. To the best of our knowledge, there is no reported in vivo FCS study on intact biological tissues. At the same time, it was found in the course of the current grant supported research that there is no existing data for determining the diffusion coefficient for Dpp in wing imaginal disc. In principle, FCS measurements should be capable of determining rates of diffusion and concentration in biological tissues. In  ADDIN EN.CITE Wang20042290Wang, Z., Marcu, O., Berns, M.W. and Marsh, J. L.2004In vivo FCS measurements of ligand diffusion in intact tissueJ. of Biomedical Optics9(2)(Wang 2004), we demonstrate that FCS is in fact feasible for studying the properties of molecules in intact Drosophila wing imaginal disc tissues. We do this by conducting FCS measurements of morphogen Dpp-EGFP fusion protein in intact Drosophila imaginal discs with two-photon excitation at 850 nm. We observed Brownian motion of Dpp morphogen within the gradient band. In addition, the analysis of the measured autocorrelation functions with a two-species model indicates that a fraction of the Dpp-EGFP is involved in a larger complex or a possible anomalous diffusion along the cell membranes. Our studies demonstrate for the first time that FCS is capable of investigating the molecular dynamics in intact tissues. Publications Supported by NIH Grant R01GM067247 (for competitive renewal): [1] Kao, J.C., Nie, Q., Teng, A., Wan, F.Y.M., Lander, A.D. and Marsh, J.L., Can Morphogen Activity Be Enhanced by Its Inhibitors? Proc. 2nd MIT Conference on Computational Mechanics (Cambridge, June), 2003, Pgs 1729-1734. [2] Lou, Y., Nie, Q. and Wan, F.Y.M., Nonlinear eigenvalue problems in the stability analysis of morphogen gradients, Studies in Appl. Math. Vol. 113, 2004, 183-215. [3] Lander, A.D., Nie, Q. and Wan, F.Y.M., Spatially Distributed Morphogen Production and Morphogen Gradient Formation, Math. Biosci. & Eng. (MBE) 2, 2005, 239 262. [4]. Lander, A.D., Nie, Q., Vargas, B. and Wan, F.Y.M., Aggregation of a distributed ligand source and morphogen gradients, Studies in Appl. Math. 114, 2005, 343 374. [5] Mizutani, C.M., Nie, Q., Wan, F.Y.M., Zhang, Y.T., Vilmos, P., Bier, E., Marsh, J.L. and Lander, A.D., Formation of MP activity gradient in the Drosophila embryo, Dev. Cell 8 (No.6), 2005, 915 924. [6] Lou, Y., Nie, Q. and Wan, F.Y.M., Effects of Sog on Dpp-receptor binding, SIAM J. Appl. Math., 65, 1748 1771. [7] Lander, A.D., Nie, Q. and Wan, F.Y.M., Internalization and end flux in morphogen gradient formation, J. of Comp. and Appl. Math. (CAM), to appear. [8] Lander, A.D., Nie, Q. and Wan, F.Y.M., Membrane associated non-receptors and morphogen gradients, Bulletin of Math. Bio., to appear. [9] Wang, Z., Marcu, O., Berns, M. W., and Marsh, J. L. (2004b). In vivo FCS measurements of ligand diffusion in intact tissues. Multiphoton Microscopy in the Biomedical Sciences IV, Proceedings of SPIE 5323, 177-183. (Also, Journal Biomedical Optics 9. In Press.) [10] Lander, A.D., Wan, F.Y.M., Elledge, H.M., Claudia Mieko Mizutani, C.M., Bier, E. and Nie, Q., Diverse Paths to Morphogen Gradient Robustness, submitted to PLoS Biology. References  QUOTE EN.REFLIST Cadigan, K. M., Fish, M.P., Rulifson, E.J., and Nusse, R. (1998). "Wingless repression of Drosoplila frizzled 2 expression shapes the Wingless morphogen gradient in the wing." Cell 93: 767-777. Harland, R. M. (2001). "Developmental biology A twist on embryonic signalling." Nature 410(6827): 423-4. Jackson, S. M., H. Nakato, et al. (1997). "division abnormally delayed, a Drosophila glypican, controls cellular responses to the TGF--related morphogen, Dpp." Development 124: 4113-4120. Lander, A., Nie, Q. , Wan, F. (2005c). "Internalization and End Flux in Morphogen Gradient Formation." Accepted, J. of Computational and Applied Math. Lander, A., Nie, Q. , Vargas, B. Wan, F. (2005b). "Aggregation of Distributed Sources in Morphogen Gradients Formation." Studies in Applied Mathematics 114(4): 343-374. Lander, A., Nie,Q. , Wan, F. (2002). "Do morphogen gradients arise by diffusion." Development Cell 2(6): 785-796. Lander, A. D., Nie, Q. , Wan, F.Y.M. (2005a). "Spatially Distributed Morphogen Production and Morphogen Gradient Formation." Mathematical Biosciences and Engineering 2: 239-262. Lander, A. D., Nie, Q. and Wan, F.Y.M. (2005d). "Membrane associated non-recptors and morphogen gradients, Bulletin of Math." Bulletin of Mathematical Biology Accepted for Publication ( HYPERLINK "http://www.math.uci.edu/~fwan/pdf/BulletinMB.pdf)" http://www.math.uci.edu/~fwan/pdf/BulletinMB.pdf). Lander, A. D., Wan, F.Y.M., Heather M. Elledge, H.M., Claudia Mieko Mizutani, C.M., Bier, E. and Nie, Q. (2005e). "Diverse Paths to Morphogen Gradient Robustness." Submitted for publication ( HYPERLINK "http://www.math.uci.edu/~fwan/pdf/DivPaths.pdf)" http://www.math.uci.edu/~fwan/pdf/DivPaths.pdf). Lander, A. N., Q. , Wan, F., Xu, J. (2003). Diffusion and Morphogen Gradient Formation. Submitted ( HYPERLINK "http://math.uci.edu/~fwan/pdf/all.pdf):" http://math.uci.edu/~fwan/pdf/all.pdf): 66 pages. Lecuit, T. and S. M. Cohen (1998). "Dpp receptor levels contribute to shaping the Dpp morphogen gradient in the Drosophila wing imaginal disc." Development 125(24): 4901-7. Axis formation in the Drosophila wing depends on the localized expression of the secreted signaling molecule Decapentaplegic (Dpp). Dpp acts directly at a distance to specify discrete spatial domains, suggesting that it functions as a morphogen. Expression levels of the Dpp receptor thick veins (tkv) are not uniform along the anterior- posterior axis of the wing imaginal disc. Receptor levels are low where Dpp induces its targets Spalt and Omb in the wing pouch. Receptor levels increase in cells farther from the source of Dpp in the lateral regions of the disc. We present evidence that Dpp signaling negatively regulates tkv expression and that the level of receptor influences the effective range of the Dpp gradient. High levels of tkv sensitize cells to low levels of Dpp and also appear to limit the movement of Dpp outside the wing pouch. Thus receptor levels help to shape the Dpp gradient. Lou, Y., Nie, Q., Wan, F. (2004). "Nonlinear Eigenvalue Problems in the Stability Analysis of Morphogen Gradients." Studies in Applied Mathematics 113: 183-215. Lou, Y., Nie, Q., Wan, F. (2005). "Effects of Sog on Dpp-Receptor Binding." SIAM J. Appl. Math. ( HYPERLINK "http://math.uci.edu/~fwan/download/LNW-SIAM.pdf" http://math.uci.edu/~fwan/download/LNW-SIAM.pdf) 65(5): 1748 - 1771. Marcu, O., Agrawal, N. , Heslip, T. and Marsh, J.L. (2005). " Syndecan modulates the distribution and signaling of Wingless morphogen in the Drosophila wing margin." Preprint ( HYPERLINK "http://www.math.uci.edu/~fwan/sdc-wg.doc/html)" http://www.math.uci.edu/~fwan/sdc-wg.doc/html). Mizutani, C., Nie,Q.,Wan, F.,Zhang, Y. ,Vilmos, P. ,Bier, E., Marsh,L., and Lander, A. (2005). "Formation of the BMP activity gradient in the Drosophila embryo." Developmental Cell 8(June-6): 1 - 10. Ray, R. P. and K. A. Wharton (2001). "Twisted Perspective. New Insights into Extracellular Modulation of BMP Signaling during Development." Cell 104(6): 801-804. Ross, J. J., O. Shimmi, et al. (2001). "Twisted gastrulation is a conserved extracellular BMP antagonist." Nature 410(6827): 479-83. Bone morphogenetic protein (BMP) signalling regulates embryonic dorsal- ventral cell fate decisions in flies, frogs and fish. BMP activity is controlled by several secreted factors including the antagonists chordin and short gastrulation (SOG). Here we show that a second secreted protein, Twisted gastrulation (Tsg), enhances the antagonistic activity of Sog/chordin. In Drosophila, visualization of BMP signalling using anti-phospho-Smad staining shows that the tsg and sog loss-of- function phenotypes are very similar. In S2 cells and imaginal discs, TSG and SOG together make a more effective inhibitor of BMP signalling than either of them alone. Blocking Tsg function in zebrafish with morpholino oligonucleotides causes ventralization similar to that produced by chordin mutants. Co-injection of sub-inhibitory levels of morpholines directed against both Tsg and chordin synergistically enhances the penetrance of the ventralized phenotype. We show that Tsgs from different species are functionally equivalent, and conclude that Tsg is a conserved protein that functions with SOG/chordin to antagonize BMP signalling. The, I., Y. Bellaiche, et al. (1999). "Hedgehog movement is regulated through tout velu-dependent synthesis of a heparan sulfate proteoglycan." Mol Cell 4(4): 633-9. Hedgehog (Hh) molecules play critical roles during development as a morphogen, and therefore their distribution must be regulated. Hh proteins undergo several modifications that tether them to the membrane. We have previously identified tout velu (ttv), a homolog of the mammalian EXT tumor suppressor gene family, as a gene required for movement of Hh. In this paper, we present in vivo evidence that ttv is involved in heparan sulfate proteoglycan (HSPG) biosynthesis, suggesting that HSPGs control Hh distribution. In contrast to mutants in other HSPG biosynthesis genes, the activity of the HSPG-dependent FGF and Wingless signaling pathways are not affected in ttv mutants. This demonstrates an unexpected level of specificity in the regulation of the distribution of extracellular signals by HSPGs. Wang, Z., Marcu, O., Berns, M.W. and Marsh, J. L. (2004). "In vivo FCS measurements of ligand diffusion in intact tissue." J. of Biomedical Optics 9(2).      PAGE  PAGE 8 intensity  EMBED Photoshop.Image.7 \s  2 1 syndecan overproduction (2) 0 normal levels of syndecan (1) Figure 6: The cell fates along the D/V axis of a fly embryo are shown. A: a micrograph of a fly embryo showing the dorsal-most amnioserosa (dark blue arrow), the dorsal-lateral ectoderm (light blue arrow) and the ventral ectoderm (red arrow). B: These domains are shown diagramatically and C in cross section. Dorsal is up and ventral down. D: shows the same cell fates with the approximate distribution of several key proteins shown. Sog (deep purple) is distributed in a gradient from ventral to dorsal. Dpp (pink) is graded from dorsal to ventral. The peak of Dpp signaling that forms is shown in green. distance across the dorso-ventral axis Figure 7. Equations describing the distribution and binding interactions of proteins in the Dpp pathway. The general form of the equations follows those outlined in 1a. Symbols are Ligand (Dpp); Tsg, (Tsg); Sog; Receptor; Tld. Tolloid. (Tld) is a secreted protease the can cleave Sog when in a complex with other factors.  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