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The classic book "On Growth and Form" by naturalist D''Arcy Thompson was published 100 years ago. To celebrate this landmark, we present experiments in the Xenopus embryo that provide a framework for understanding how simple, quantitative transformations of a morphogen gradient might have affected evolution and morphological diversity of organisms. D''Arcy Thompson proposed that different morphologies might be generated by modifying physical parameters in an underlying system of Cartesian coordinates that pre-existed in Nature and arose during evolutionary history. Chordin is a BMP antagonist secreted by the Spemann organizer located on the dorsal side of the gastrula. Chordin generates a morphogen gradient as first proposed by mathematician Alan Turing. The rate-limiting step of this dorsal-ventral (D-V) morphogen is the degradation of Chordin by the Tolloid metalloproteinase in the ventral side. Chordin is expressed at gastrula on the dorsal side where BMP signaling is low, while at the opposite side peak levels of BMP signaling are reached. In fishes, amphibians, reptiles and birds, high BMP signaling in the ventral region induces transcription of a secreted inhibitor of Tolloid called Sizzled. By depleting Sizzled exclusively in the ventral half of the embryo we were able to expand the ventro-posterior region in an otherwise normal embryo. Conversely, ventral depletion of Tolloid, which stabilizes Chordin, decreased ventral and tail structures, phenocopying the tolloid zebrafish mutation. We explain how historical constraints recorded in the language of DNA become subject to the universal laws of physics when an ancestral reaction-diffusion morphogen gradient dictates form.
D'Arcy Thompson's 1917 illustration of his theory of coordinate transformations during evolution of related animals. In his view, morphological change occurs through transformations of the animal body plan within the constraints of a set of Cartesian coordinates. This is shown here for two closely related fish species, the porcupine fish Diodon (left) and the sunfish Orthagoricus (right, also known as Mola mola). We propose that modification of the Chordin/Tolloid/BMP morphogen gradient could have mediated such evolutionary changes in body forms.
The Chordin/Tolloid/BMP morphogenetic pathway. This pathway patterns the D-V axis of the Xenopus gastrula. A self-regulating gradient of BMP activity results from direct protein–protein interactions between Chordin and other partners, indicated by black lines. Blue arrows indicate transcriptional regulation by BMP signaling, which is maximal in the ventral and lowest in the dorsal center. Red arrows indicate protein flux of Chordin-BMP complexes in a narrow signaling highway located in the extracellular matrix that separates the ectoderm from the endomesoderm. Tolloid constitutes the rate-limiting step, serving as a Chordin proteinase. Note that BMPs have a dual regulatory activity on Tolloid metalloproteinase: they stimulate Tolloid (Xlr) transcription, and they inhibit Tolloid enzymatic activity non-competitively by binding to the CUB domains. Sizzled is a competitive inhibitor of Tolloid that is bound by the active site but not cleaved.
Depletion of Sizzled on the ventral side results in embryos with expanded ventral-posterior structures and normal heads. (A) Schematic diagram showing targeted injection of Sizzled antisense Morpholino into the ventral blastomeres of a four-cell Xenopus embryo. (B,C) Injected and control embryos were collected at stage 25 and analyzed for phenotype; five independent experiments. All controls (n = 150) developed normally. Ventrally targeted Sizzled depletion promoted enlargement of ventro-posterior tissues, while leaving the anterior tissues unaffected. This phenotype was observed in 94% of the Szl-MO injected embryos (n = 117). Compare to Figure 1.
Depletion of Xenopus Tolloids with antisense morpholino oligos results in embryos with reduced ventral posterior structures. Note that this phenotype is the opposite of that of depletion of Sizzled. (A) Schematic diagram showing targeted injection of Tolloid MO mixtures into the ventral blastomeres of a four-cell Xenopus embryo. Embryos were analyzed at late tailbud stages (stage 34 onward) in at least five independent experiments; embryos that did not survive until tailbud were discarded and not scored. (B,D) Control embryos developed normally (n = 163). (C) 66% of the embryos (n = 53) injected with a triple mixture of Tolloid Morpholinos (BMP1/Xlr/Xld-MO) developed posterior defects with shorter tail and reduced ventral fin (which indicates lower BMP signaling). (E) A double mixture of BMP1 and Xlr MOs produced results with similar penetrance (67%, n = 68); shown here is the most affected embryo, with a severe truncation of the tail. (F,G) The Xenopus results phenocopy the minifin mutation in zebrafish, in which the homologous Tolloid gene is inactivated and leads to tail defects (drawing based on results from Connors et al. 1999).
Example of a partial differential equation of the type formulated by Alan Turing in 1952 to describe the changes in morphogen concentration (δC) over time (δt). The right side of the equation shows that, following Fick's law of diffusion, the change in concentration of a morphogen at any given point is proportional to its diffusion rate and to the second derivative in space of morphogen concentration. In addition, the change in morphogen concentration is also a function (F) of all the chemical reactions it undergoes. This insight brought physics, chemistry and mathematics into developmental biology.
The Chordin/Tolloid/BMP biochemical pathway is conserved in many different animal phyla. Although the core signaling mechanism is ancestral to many organisms, its flexibility to regulatory changes provide a molecular pattern to generate a myriad animal forms. The homologue of Chordin is called Sog in some animals, and BMP called Dpp. A Chordin/Tolloid/Dpp system was already present in the sea anemone Nematostella, a primitive diploblast (two germ layers). This D-V patterning system is dedicated to generating a BMP signaling gradient spanning the entire gastrula embryo. Illustration modified and reproduced from Bier & De Robertis (2015) with permission from AAAS Publishing. Copyright 2015. Sketches of organisms by Dr.Valentino Gantz are gratefully acknowledged.