THE FEBS ⁄ EMBO WOMEN IN SCIENCE LECTURE ‘Big frog, small frog’ – maintaining proportions in embryonic development Delivered on July 2008 at the 33rd FEBS Congress in Athens, Greece Naama Barkai and Danny Ben-Zvi Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel Keywords Admp; BMP; Chordin; control theory; development; dorsal-ventral; feedback; morphogen gradient; scaling; Xenopus Correspondence N Barkai, Department of Molecular Genetics, Weizmann Institute of Science, PO Box 26, Rehovot 76100, Israel Fax: +972 934 4108 Tel: +972 934 4429 E-mail: naama.barkai@weizmann.ac.il (Received 10 October 2008, revised December 2008, accepted 11 December 2008) We discuss mechanisms that enable the scaling of pattern with size during the development of multicellular organisms Recently, we analyzed scaling in the context of the early Xenopus embryo, focusing on the determination of the dorsal–ventral axis by a gradient of BMP activation The ability of this system to withstand extreme perturbation was exemplified in classical experiments performed by Hans Spemann in the early 20th century Quantitative analysis revealed that patterning is governed by a noncanonical ‘shuttling-based’ mechanism, and defined the feedback enabling the scaling of pattern with size Robust scaling is due to molecular implementation of an integral-feedback controller, which adjusts the width of the BMP morphogen gradient with the size of the system We present an ‘expansion– repression’ feedback topology which generalizes this concept for a wider range of patterning systems, providing a general, and potentially widely applicable model for the robust scaling of morphogen gradients with size doi:10.1111/j.1742-4658.2008.06854.x Spemann’s experiments and the scaling of pattern with size in the amphibian embryo From the early days of embryology, biologists have marveled at the remarkable consistency of the developing body plan In 1942, Conrad Waddington put forward the concept of canalization, referring to the invariance of the wild-type phenotype in the face of genetic or environmental perturbations [1] Since then, extensive research has been devoted to understand the origins and evolutionary implications of this fundamental property of developing organisms [2] The plasticity of embryonic development, with its ability to overcome extreme perturbations, was demonstrated most dramatically in two classic experiments performed by Hans Spemann at the beginning of the 20th century [3–5] (Fig 1A,B) In 1903, Spemann used a thin baby hair to bisect a cleaving newt embryo into dorsal and ventral halves Remarkably, dorsal-halved, but not ventral-halved, embryos healed and developed into normal, albeit smaller tadpoles Twenty years later, in 1924, Hilde Mangold joined Spemann to perform a second fascinating experiment in which they transplanted a group of dorsal cells (‘dorsal lip’) grafted from a donor embryo into the ventral pole of a recipient embryo Strikingly, a complete secondary axis ensued, resulting in Siamese twins The transplanted cells re-specified host tissues to form neural tissues and somites instead of epidermis and ventral– posterior mesoderm The transplanted cells themselves only contributed to a fraction of the secondary axis, Abbreviations Admp, anti-dorsalizing morphogenic protein; BMP, bone morphogenic protein 1196 FEBS Journal 276 (2009) 1196–1207 ª 2009 The Authors Journal compilation ª 2009 FEBS N Barkai and D Ben-Zvi Maintaining proportions in embryonic development Fig Scaling of the BMP gradient along the dorsal–ventral axis in Xenopus embryos (following Reversade & De Robertis [27]) (A, B) The Spemann experiments (A) The dorsal half of a Xenopus embryo has the capacity to develop into a complete, though smaller complete embryo, whereas the ventral half develops into a ‘bellypiece’ (B) When the Spemann Organizer, located at the dorsal–vegetal side of a donor Xenopus embryo, is transplanted into the ventral–vegetal side of a recipient embryo (arrowhead), two complete axes ensue (C) Schematic vegetal view of the Xenopus embryo at early gastrula chordin and admp are expressed on the dorsal side, and bmp4 is expressed over a wide region centered on the ventral side The BMP signaling gradient ranges from blue (high) to red (low) (D) The problem of scaling morphogen gradients The BMP signaling gradient is can induce at least four cell fates along the dorsal–ventral morphogenic field (0 < x < L, upper) If the field is shorter (0 < x 0: ð4Þ dE Here bE > is the rate by which E is produced per unit length, and a0 is some dimensionless constant Fig Expansion–repression mechanism (A) Expansion–repression feedback is based on two properties First, the morphogen represses an expander molecule Second, the expander functions to increases the spread of the morphogen, k, by some mechanism such as enhancing morphogen diffusion or reducing its degradation The expander must be diffusible and relatively stable (B) An integral feedback controller underlies the scaling mechanism The target of the control circuit is to scale the gradient with the size of the field The morphogen gradient (system output) is measured by induction ⁄ repression of the expander in each cell (sensor) xrep, the distal most position where the expander is induced (measure error) is compared with the desired scale, for which the expander is not induced at all, i.e xrep = L (reference) The region where the expander is induced (measured error) produces the expander, which accumulates in the field This accumulation turns the controller into an integral controller The increase in the expander level (system input) increases the length scale of the gradient (system) This increase changes the morphogen gradient (system output), and the process is repeated with the induction ⁄ repression of the expander This process halts when xrep equals the distal-most position in the field, hence the expander levels and length scale stabilize (C) Schematic representation of expansion–repression dynamics High morphogen signaling in shown in green, whereas low signaling is shown in red The morphogen is produced and secreted at the proximal region Initially, its spread is small and the gradient is narrow Consequently, the expander (purple) is expressed and is secreted over a wide area in the distal region of the field (upper) Accumulation and diffusion of the Expander expands the gradient (middle) until the gradient is wide enough to repress the expander everywhere in the field (lower) The expander may interact with the heparan sulfate proteoglycans, receptors or any other elements to increase the spread of the gradient 1202 FEBS Journal 276 (2009) 1196–1207 ª 2009 The Authors Journal compilation ª 2009 FEBS N Barkai and D Ben-Zvi FEBS Journal 276 (2009) 1196–1207 ª 2009 The Authors Journal compilation ª 2009 FEBS Maintaining proportions in embryonic development 1203 Maintaining proportions in embryonic development N Barkai and D Ben-Zvi independent of the field size, L The directionality of the feedback ensures that at a steady state, E is properly adjusted such that L = a0kst, implying scaling of the morphogen profile with the size of the field Clearly, these equations define an integral-feedback controller Thus, any implementation of the ‘expansion–repression’ feedback module, regardless of the exact molecular details, will lead to robust scaling of the morphogen pattern with the size of the field Comparison with other scaling mechanisms The main advantage of the ‘expansion–repression’ mechanism for scaling is its robustness The use of integral-feedback ensures scaling for a wide range of parameters without the need to fine-tune rate constants or the precise functional dependency between the different parameters Scaling is achieved by the structure of the network, independent of other aspects of the morphogen gradient such as degradation and transport mechanisms Previous theoretical attempts to explain scaling have focused on three general paradigms, described below Arguably, the simplest scaling mechanism is the so-called ‘perfect sink’ solution A ‘perfect sink’ degrades morphogen rapidly, and consumes all morphogen molecules reaching its position If positioned at the edge of the field, opposing the morphogen source, a perfect sink will lead to scaling, but only when (a) the morphogen does not degrade during its motion within the field and (b) morphogen levels at the source are kept constant Biologically, these two conditions rarely hold In most cases the morphogen does degrade during its movement across the tissue through interaction with inhibitors or with receptors Moreover, it is probably the rate of production at the source, rather then the level of morphogen, which is kept fixed It is therefore unlikely that perfect sink contributes to scaling in most biologically relevant situations Several studies have suggested that scaling is achieved through the integration of two opposing gradients, e.g when two morphogen sources are positioned at two opposing poles [62–64] In this case, cells can extract information about the size of the field by effectively comparing the two gradients If the morphogen degrades linearly, scaling is guaranteed for a single position within the field This mechanism cannot be used to scale multiple threshold positions, in sharp contrast to the situations described above for the ‘expansion–repression’ topology, where nearly all threshold positions scaled with the size of the field, maintaining proportions The situation somewhat improves if both morphogens are 1204 degraded in the exact same nonlinear manner throughout the field In this case, scaling holds over a wide domain of the field (N Barkai & D Ben-Zvi, unpublished results) However, even under these conditions, scaling requires the ‘fine-tuning’ of the reactions of the two molecular gradients, a fact which might limit its biological application The ‘expansion–repression’ feedback is more related to a third class of mechanisms, which assumes the existence of a chemical species, analogous to the proposed ‘expander’, whose concentration affects the length scale (spread) of the morphogen gradient In the context of self-organized patterning (‘turing-like’ mechanism), a similar chemical species alters the wavelength of the activator profile [65–67] The level of this secreted species is assumed to be proportional to some power of the field size, depending on specific assumptions and boundary conditions No feedback, however, is assumed between the morphogen signaling and the production of this secreted species The proposed ‘expansion–repression’ feedback topology thus extends and generalizes this approach, by introducing feedback on the production of the expander molecule and applying it upon the standard morphogen gradient paradigm This feedback results in an effective integral-feedback controller, enabling a robust scaling of morphogen spread with the system size, in a manner that does not depend on parameters or on the details of the interactions in the system Other successful attempts to model scaling in specific systems [68,69] considered scaling of a single position of the field and not the entire gradient, and relied on the unique properties of those systems Concluding remarks The development of multicellular organisms is characterized by extensive changes in size and morphology Growth and patterning must be coordinated, and the ability to scale pattern with size is one manifestation of this coordination Coordination can be achieved if size is defined by the patterning process itself, e.g if tissue size is controlled by precisely the same morphogen gradient that defines tissue pattern External factors governing size may also take effect through changing the physical properties and the length scale of the morphogen gradient Alternatively, size can be defined independently, and scaling of pattern achieved at the level of the patterning process itself This review has focused on the latter paradigm, which appears to hold for early developmental processes It is possible that later processes are governed by a more intricate interplay between growth and patterning FEBS Journal 276 (2009) 1196–1207 ª 2009 The Authors Journal compilation ª 2009 FEBS N Barkai and D Ben-Zvi The scaling mechanism we describe implements, in molecular terms, the concept of integral-feedback control The main advantage of this mechanism is its robustness: scaling does not require ‘fine-tuning’ of reaction rate constants but is inherent to the mechanism itself Moreover, there is no need for precise adjustment of the molecular interactions Scaling is the outcome of the general feedback topology, which can be implemented in a variety of ways For example, in the basic ‘expansion–repression’ topology, all that is required is for the Expander molecule to be widely diffusible and stable, to be repressed by morphogen signaling and influence (in some unspecified way) the diffusion or degradation of the morphogen This mechanism can be applied in various ways by developing organisms, as we have shown for the Xenopus embryo The ability to scale pattern with size is highly important for normal development It enables the organism to compensate for natural variation and overcome periods of nutrient limitation, which reduce embryo and tissue size In addition, such a capacity may also be important for facilitating the evolutionary adaptation of body size, because the pattern will automatically adjust with any mutation that alters body size, without the need for further adjustment of the patterning mechanism It will be interesting to examine whether the same scaling mechanisms that function within a given species, also operate to define the difference in size between species References Waddington CH (1942) canalization of development and the inheritance of acquired characters Nature 150, 563 Braendle C & Felix MA (2008) Plasticity and errors of a robust developmental system in different environments Dev Cell 15, 714–724 Spemann H (1938) Embryonic Development and Induction Yale University Press, New Haven, CT Spemann H & Mangold H (1924) Induction of embryonic primordia by implantation of organizers from a different species Roux’s Arch Entw Mech 100, 599– 638 De Robertis EM (2006) Spemann’s organizer and selfregulation in amphibian embryos Nat Rev Mol Cell Biol 7, 296–302 Bertocchini F, Skromne I, Wolpert L & Stern CD (2004) Determination of embryonic polarity in a regulative system: evidence for endogenous inhibitors acting sequentially during primitive streak formation in the chick embryo Development 131, 3381–3390 Maintaining proportions in embryonic development Joubin K & Stern CD (1999) Molecular interactions continuously define the organizer during the cell movements of gastrulation Cell 98, 559–571 Enders AC (2002) Implantation in the nine-banded armadillo: how does a single blastocyst form four embryos? Placenta 23, 71–85 Sadler TW (2004) Langman’s Medical Embryology Lippincott Williams & Wilkins, Philadelphia 10 Cooke J (1981) Scale of body pattern adjusts to available cell number in amphibian embryos Nature 290, 775–778 11 Gerhart J, Lowe C & Kirschner M (2005) Hemichordates and the origin of chordates Curr Opin Genet Dev 15, 461–467 12 Lowe CJ, Terasaki M, Wu M, Freeman RM Jr, Runft L, Kwan K, Haigo S, Aronowicz J, Lander E, Gruber C et al (2006) Dorsoventral patterning in hemichordates: insights into early chordate evolution PLoS Biol 4, e291 13 Ferguson EL (1996) Conservation of dorsal–ventral patterning in arthropods and chordates Curr Opin Genet Dev 6, 424–431 14 Mieko Mizutani C & Bier E (2008) EvoD ⁄ Vo: the origins of BMP signalling in the neuroectoderm Nat Rev Genet 9, 663–677 15 De Robertis EM & Kuroda H (2004) Dorsal–ventral patterning and neural induction in Xenopus embryos Annu Rev Cell Dev Biol 20, 285–308 16 Harland RM (1994) Neural induction in Xenopus Curr Opin Genet Dev 4, 543–549 17 Dosch R, Gawantka V, Delius H, Blumenstock C & Niehrs C (1997) Bmp-4 acts as a morphogen in dorsoventral mesoderm patterning in Xenopus Development 124, 2325–2334 18 Kurata T, Nakabayashi J, Yamamoto TS, Mochii M & Ueno N (2001) Visualization of endogenous BMP signaling during Xenopus development Differentiation 67, 33–40 19 Schohl A & Fagotto F (2002) Beta-catenin, MAPK and Smad signaling during early Xenopus development Development 129, 37–52 20 Ben-Zvi D, Shilo BZ, Fainsod A & Barkai N (2008) Scaling of the BMP activation gradient in Xenopus embryos Nature 453, 1205–1211 21 Blitz IL, Shimmi O, Wunnenberg-Stapleton K, O’Connor MB & Cho KW (2000) Is chordin a long-range- or short-range-acting factor? Roles for BMP1-related metalloproteases in chordin and BMP4 autofeedback loop regulation Dev Biol 223, 120–138 22 Hama J & Weinstein DC (2001) Is chordin a morphogen? Bioessays 23, 121–124 23 Dale L & Jones CM (1999) BMP signalling in early Xenopus development Bioessays 21, 751–760 FEBS Journal 276 (2009) 1196–1207 ª 2009 The Authors Journal compilation ª 2009 FEBS 1205 Maintaining proportions in embryonic development N Barkai and D Ben-Zvi 24 Hawley SH, Wunnenberg-Stapleton K, Hashimoto C, Laurent MN, Watabe T, Blumberg BW & Cho KW (1995) Disruption of BMP signals in embryonic Xenopus ectoderm leads to direct neural induction Genes Dev 9, 2923–2935 25 Dosch R & Niehrs C (2000) Requirement for anti-dorsalizing morphogenetic protein in organizer patterning Mech Dev 90, 195–203 26 Moos M, Wang S & Krinks M (1995) Anti-dorsalizing morphogenetic protein is a novel TGF-beta homolog expressed in the Spemann organizer Development 121, 4293–4301 27 Reversade B & De Robertis EM (2005) Regulation of ADMP and BMP2 ⁄ ⁄ at opposite embryonic poles generates a self-regulating morphogenetic field Cell 123, 1147–1160 28 Little SC & Mullins MC (2006) Extracellular modulation of BMP activity in patterning the dorsoventral axis Birth Defects Research Part C, Embryo Today: Reviews 78, 224–242 29 Lee HX, Ambrosio AL, Reversade B & De Robertis EM (2006) Embryonic dorsal–ventral signaling: secreted frizzled-related proteins as inhibitors of tolloid proteinases Cell 124, 147–159 30 Inomata H, Haraguchi T & Sasai Y (2008) Robust stability of the embryonic axial pattern requires a secreted scaffold for chordin degradation Cell 134, 854–865 31 Ambrosio AL, Taelman VF, Lee HX, Metzinger CA, Coffinier C & De Robertis EM (2008) Crossveinless-2 is a BMP feedback inhibitor that binds chordin ⁄ BMP to regulate Xenopus embryonic patterning Dev Cell 15, 248–260 32 Eldar A, Dorfman R, Weiss D, Ashe H, Shilo BZ & Barkai N (2002) Robustness of the BMP morphogen gradient in Drosophila embryonic patterning Nature 419, 304–308 33 Meinhardt H & Roth S (2002) Developmental biology: sharp peaks from shallow sources Nature 419, 261–262 34 Mizutani CM (2005) Formation of the BMP activity gradient in the Drosophila embryo Dev Cell 8, 915–924 35 Shimmi O, Umulis D, Othmer H & O’Connor MB (2005) Facilitated transport of a Dpp ⁄ Scw heterodimer by Sog ⁄ Tsg leads to robust patterning of the Drosophila blastoderm embryo Cell 120, 873–886 36 Wang YC & Ferguson EL (2005) Spatial bistability of Dpp-receptor interactions during Drosophila dorsal– ventral patterning Nature 434, 229–234 37 Zee Mvd, Stockhammer O, Levetzow Cv, Fonseca RNd & Roth S (2006) Sog ⁄ Chordin is required for ventralto-dorsal Dpp ⁄ BMP transport and head formation in a short germ insect Proc Natl Acad Sci USA 103, 16307– 16312 38 O’Connor MB, Umulis D, Othmer HG & Blair SS (2006) Shaping BMP morphogen gradients in the 1206 39 40 41 42 43 44 45 46 47 48 49 50 51 52 Drosophila embryo and pupal wing Development 133, 183–193 Yi TM, Huang Y, Simon MI & Doyle J (2000) Robust perfect adaptation in bacterial chemotaxis through integral feedback control Proc Natl Acad Sci USA 97, 4649–4653 Hufnagel L, Kreuger J, Cohen SM & Shraiman BI (2006) On the role of glypicans in the process of morphogen gradient formation Dev Biol 300, 512– 522 Cooke J (1975) Control of somite number during morphogenesis of a vertebrate, Xenopus laevis Nature 254, 196–199 Dzialowski EM & Sotherland PR (2004) Maternal effects of egg size on emu Dromaius novaehollandiae egg composition and hatchling phenotype J Exp Biol 207, 597–606 Phillips NE (2007) High variability in egg size and energetic content among intertidal mussels Biol Bull 212, 12–19 Smith RC & Neff AW (1986) Organisation of Xenopus egg cytoplasm: response to simulated microgravity J Exp Zool 239, 365–378 Driesch H (1891) Der Werth der beiden ersten Furchungszellen in der Echinodermentwicklung Experimentelle Erzeugung von Theil und Doppelbildungen Z Wiss Zool 53, 160–178 Morgan TH (1895) Half embryos and whole embryos from one of the first two blastomeres Anat Anz 10, 623–638 Bryant PJ & Simpson P (1984) Intrinsic and extrinsic control of growth in developing organs Q Rev Biol 59, 387–415 Nijhout HF & Emlen DJ (1998) Competition among body parts in the development and evolution of insect morphology Proc Natl Acad Sci USA 95, 3685–3689 Bohni R, Riesgo-Escovar J, Oldham S, Brogiolo W, Stocker H, Andruss BF, Beckingham K & Hafen E (1999) Autonomous control of cell and organ size by CHICO, a Drosophila homolog of vertebrate IRS1-4 Cell 97, 865–875 Leevers SJ, Weinkove D, MacDougall LK, Hafen E & Waterfield MD (1996) The Drosophila phosphoinositide 3-kinase Dp110 promotes cell growth EMBO J 15, 6584–6594 Montagne J, Stewart MJ, Stocker H, Hafen E, Kozma SC & Thomas G (1999) Drosophila S6 kinase: a regulator of cell size Science 285, 2126–2129 Weinkove D, Neufeld TP, Twardzik T, Waterfield MD & Leevers SJ (1999) Regulation of imaginal disc cell size, cell number and organ size by Drosophila class I(A) phosphoinositide 3-kinase and its adaptor Curr Biol 9, 1019–1029 FEBS Journal 276 (2009) 1196–1207 ª 2009 The Authors Journal compilation ª 2009 FEBS N Barkai and D Ben-Zvi 53 Kuzin B, Roberts I, Peunova N & Enikolopov G (1996) Nitric oxide regulates cell proliferation during Drosophila development Cell 87, 639–649 54 Day SJ & Lawrence PA (2000) Measuring dimensions: the regulation of size and shape Development 127, 2977–2987 55 Teleman AA & Cohen SM (2000) Dpp gradient formation in the Drosophila wing imaginal disc Cell 103, 971–980 56 Goberdhan DC, Paricio N, Goodman EC, Mlodzik M & Wilson C (1999) Drosophila tumor suppressor PTEN controls cell size and number by antagonizing the Chico ⁄ PI3-kinase signaling pathway Genes Dev 13, 3244–3258 57 Cadigan KM (2002) Regulating morphogen gradients in the Drosophila wing Semin Cell Dev Biol 13, 83–90 58 Dessaud E, McMahon AP & Briscoe J (2008) Pattern formation in the vertebrate neural tube: a sonic hedgehog morphogen-regulated transcriptional network Development 135, 2489–2503 59 Akiyama T, Kamimura K, Firkus C, Takeo S, Shimmi O & Nakato H (2008) Dally regulates Dpp morphogen gradient formation by stabilizing Dpp on the cell surface Dev Biol 313, 408–419 60 Tsuda M, Kamimura K, Nakato H, Archer M, Staatz W, Fox B, Humphrey M, Olson S, Futch T, Kaluza V et al (1999) The cell-surface proteoglycan Dally regulates Wingless signalling in Drosophila Nature 400, 276–280 Maintaining proportions in embryonic development 61 Chen Y & Struhl G (1996) Dual roles for patched in sequestering and transducing Hedgehog Cell 87, 553– 563 62 Houchmandzadeh B, Wieschaus E & Leibler S (2005) Precise domain specification in the developing Drosophila embryo Phys Rev E Stat Nonlin Soft Matter Phys 72, 061920 63 McHale P, Rappel WJ & Levine H (2006) Embryonic pattern scaling achieved by oppositely directed morphogen gradients Phys Biol 3, 107–120 64 Howard M & ten Wolde PR (2005) Finding the center reliably: robust patterns of developmental gene expression Phys Rev Lett 95, 208103 65 Othmer HG & Pate E (1980) Scale-invariance in reaction-diffusion models of spatial pattern formation Proc Natl Acad Sci USA 77, 4180–4184 66 Hunding A & Sorensen PG (1988) Size adaptation of Turing prepatterns J Math Biol 26, 27–39 67 Ishihara S & Kaneko K (2006) Turing pattern with proportion preservation J Theor Biol 238, 683– 693 68 Umulis DM, Serpe M, O¢Connor MB & Othmer HG (2006) Robust, bistable patterning of the dorsal surface of the Drosophila embryo Proc Natl Acad Sci USA 103, 11613–11618 69 He F, Wen Y, Deng J, Lin X, Lu LJ, Jiao R & Ma J (2008) Probing intrinsic properties of a robust morphogen gradient in Drosophila Dev Cell 15, 558– 567 FEBS Journal 276 (2009) 1196–1207 ª 2009 The Authors Journal compilation ª 2009 FEBS 1207 ... Ben-Zvi FEBS Journal 27 6 (20 09) 119 6–1 20 7 ª 20 09 The Authors Journal compilation ª 20 09 FEBS Maintaining proportions in embryonic development 120 3 Maintaining proportions in embryonic development. .. 27 6 (20 09) 119 6–1 20 7 ª 20 09 The Authors Journal compilation ª 20 09 FEBS 1197 Maintaining proportions in embryonic development N Barkai and D Ben-Zvi primarily the notochord The embryonic region... FEBS Journal 27 6 (20 09) 119 6–1 20 7 ª 20 09 The Authors Journal compilation ª 20 09 FEBS 120 1 Maintaining proportions in embryonic development N Barkai and D Ben-Zvi occurs early, at the level of