Morphological Evolution: Epigenetic Mechanisms

Morphological Evolution: Epigenetic Mechanisms Stuart A Newman, New York Medical College, Valhalla, New York, USA Gerd B Mu¨ller, University of Vienna, Vienna, Austria

Special Essay article Article Contents . Introduction . The Physics of Multicellularity and the Origin of Body Plans . The Epigenetics of Advanced Development . The Origin of Morphological Homology

Organismal forms have not always been generated by the highly integrated genetic ‘programs’ characteristic of modern multicellular species. Physical forces and other conditional processes played a more prominent role at earlier stages of evolution, establishing morphological templates that were consolidated by later genetic change. These mechanisms are responsible for continued generation of morphological novelty, and are ultimately involved in the establishment of the individualized and heritable construction units of morphological evolution known as homologues.

Introduction Materials of the nonliving world take on forms dictated by external forces to which they are susceptible by virtue of their inherent physical properties. Water, for example, forms waves and vortices if it is mechanically agitated, while clay bears the record of its most recent physical impressions long after they have been exerted. Living metazoa – multicellular animals – seem to obey different rules: their forms appear to be expressions of intrinsic genetic ‘programs’. While organisms must, of course, exchange energy and matter with the external world to stay alive, the general plans and fine details of the forms they assume are taken to have become independent of the external environment. But consideration of the results of modern developmental biology suggests that at early stages in their evolution the forms of metazoan organisms were not generated in such a rigid programmatic fashion. Rather, the earliest multicellular organisms were almost certainly moulded by their physical environments to a much greater extent than contemporary organisms, and, with regard to the generation of three-dimensional form, were more like certain materials of the nonliving world than are their modern, highly evolved counterparts. Contemporary organisms are characterized by redundancies of gene action and highly integrated signalling networks which ensure that developmental pathways are reliable and resistant to perturbation. In contrast, the most ancient multicellular creatures must have been simple cell aggregates that arose by adhesion of originally free-living cells, or by the failure of the same to separate after mitosis. Although the single-celled progenitors were themselves the sophisticated products of a billion years or more of evolution, the steps that made them multicellular could have been as simple as a single mutation that rendered a cell surface protein sticky, or even a change in the ion content of the sea that provided a pre-existing protein with this new

property. Once this occurred, and it probably occurred more than once in the history of life, living organisms became susceptible to new sets of determinants: initially the forces that mould what physicists refer to as ‘soft matter’ and the inherent self-organizing capabilities of chemically active materials, and later other conditional form-generating processes, such as tissue inductive interactions. We refer to these conditional, ‘non-programmatic’ determinants collectively as ‘epigenetic’ mechanisms.

The Physics of Multicellularity and the Origin of Body Plans Multicellular organisms first arose more than 600 million years ago. By approximately 540 million years ago, at the end of the ‘Cambrian explosion’, virtually all the ‘bauplans’ or body types seen in modern organisms already existed. While the early world contained many unoccupied niches within which new organismal forms could flourish, this alone can neither account for the rapid profusion of body plans once multicellularity was established, nor for the particular forms that bodies and organs took on. In particular, metazoan bodies are characterized by axial symmetries and asymmetries, multiple tissue layers, interior cavities, segmentation, and various combinations of these properties. The organs of these creatures are organized in similar ways, on a smaller scale. Just as we recognize that liquids, clays, taut strings and soap bubbles can take on only limited, characteristic arrays of shapes and configurations, it is reasonable to ask what the characteristic spectrum of forms would have been for ancient multicellular ‘matter’. Such matter consisted of cells producing numerous gene products, some of which may have been released, and some of which remained at the surface, providing the means for cell aggregation. Although modern organisms contain highly integrated

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Morphological Evolution: Epigenetic Mechanisms

genetic mechanisms of pattern formation and morphogenesis, these could not have been present at the dawn of multicellularity. Rather, as will be described, the body and tissue forms we have come to associate with modern multicellular organisms were already inherent in the material make-up of their ancient, less programmed ancestors.

Diffusion and differential adhesion The advent of cell–cell adhesion early in the history of multicellular life opened up possibilities for the moulding of bodies and their tissues that were unavailable to singlecelled organisms. The primary reason for this is that different sets of physical forces predominate at different spatial scales – the shapes and forms of macroscopic objects, such as multicellular aggregates, are influenced by physical determinants different from those that noticeably affect microscopic objects, such as individual cells. Diffusion of typical biomolecules, for example, is so rapid over the distances within a single cell that in the absence of special docking or compartmentalization mechanisms intracellular molecules will be well-mixed. In contrast, on the scale of a cell aggregate, the formation of gradients of released molecules is fostered, rather than undermined, by diffusion (Crick, 1970). A group of cells in one region of an aggregate that release a product at a higher rate than their neighbours – either by a spontaneous, stochastic effect, or because they encounter something novel in the environment that induces them to do so – can take on a privileged, organizing role in the aggregate, particularly if an effect of the product is to inhibit surrounding cells from making the same thing. It is not important that the ‘organizer’ cells be preordained: once they are established they suppress other such activities, and therefore have global patterning consequences. Because each cell embodies complex biochemical circuitry, the tissues they comprise represent ‘excitable media’ (see below). In such a context simple diffusion can become integrated into ‘reaction–diffusion’ processes, and the resulting patterns will exhibit spatial periodicities and other nonuniformities (Newman, 1993). Generation of molecular gradients by diffusion-dependent processes is used widely in contemporary metazoan patterning, but the basic ingredients were present even in the most primitive cell aggregates. Another example pertains to cell adhesion itself, the basic defining condition of multicellularity. Unlike modern organisms, which have numerous highly evolved regulatory mechanisms devoted to controlling the precise strength of intercellular adhesion, the earliest cell aggregates would have been novices in the regulation of cell–cell interactions. Cell stickiness, with little evolutionary history behind it (by some accounts, it may even have arisen as a result of changes in the ionic composition of seawater), is 2

likely to have been less stringently regulated in the earliest metazoa than it is at present. But we know from experiments in which cell types with different amounts of adhesion molecules on their surfaces are mixed together that they will sort out into islands of more cohesive cells within lakes composed of their less cohesive neighbours. Eventually, by random cell movement, the islands coalesce and an interface is established, across which cells will not intermix (Steinberg, 1998). What is observed is similar to what happens when two immiscible liquids, such as oil and water, are poured into the same container. In other words, when several differentially adhesive cell populations are present within the same tissue mass (as they would have been in primitive metazoan ancestors), multilayered structures can form automatically, comprising distinct nonmixing ‘compartments’. Gastrulation, the set of developmental processes by which tissue layers and their relative positions are achieved, is a hallmark of most metazoan groups. While such behaviours appear to be goal-directed expressions of genetic programs (and are indeed largely so in modern organisms), their evolutionary origin is likely to reside in the physical consequences of nonmixing compartments of differentially adhesive cells.

Cell aggregates as excitable media Because individual cells are metabolically active, thermodynamically ‘open systems’, tissues composed of them are ‘excitable media’, defined as materials that actively respond to their environment, mechanically, chemically or electrically. Nonliving examples have been well studied. Even the most primitive cell aggregates comprised cells that were highly evolved metabolically, and therefore embodied complex biochemical and genetic networks, responsive to the external environment. The existence of positive and negative feedback loops of chemical activity, when confined to the interior of an individual cell, will often lead to temporal oscillations in one or more chemical component (Goldbeter, 1996). Oscillations of glycolytic intermediates, for example, were identified as dynamical curiosities in yeast more than 40 years ago. Functional roles for these periodic activities are obscure, and may not exist; what is clear is that they arise as spontaneous ‘side effects’ of the metabolic circuitry, rather than as the expression of an evolved program. Once cells that were capable of generating such oscillations became incorporated into aggregates, or primitive tissues, the effect of scale was again played out in terms of novel, physically determined effects. The cell division cycle is a temporally periodic process that has no morphological consequence in the world of single cells. Even in a multicellular entity it typically acts only to increase the mass of the undifferentiated aggregate. But in a



multicellular entity exhibiting a biochemical oscillation with a period different from the cell cycle, populations of cells will be periodically generated with distinct, recurrent, initial compositions. From such cellular periodicities it is only a few steps to segmental tissue organization, which can therefore be predicted to have arisen numerous times in the history of life (Newman, 1993).

Cell polarity and lumen formation The first multicellular organisms were likely to have been composed of cells with a uniform, or random, distribution of adhesive molecules on their surfaces. Many modern cell types, in contrast, are polarized, capable of allocating different molecular species to their apical and basolateral regions. The targeting of adhesive molecules, or antiadhesive molecules, to specific regions of the cell surface has dramatic consequences. A tissue mass consisting of motile cells that are nonadhesive over portions of their surfaces will readily develop cavities or lumens (Tsarfaty et al., 1994). If such spaces come to adjoin one another, as a result of random cell movement, they will readily fuse. Lumen formation could therefore have originated as a simple consequence of differential adhesion in cells that express adhesive properties in a polarized fashion. Significantly, the first morphologically complex multicellular organisms, represented by the Vendian fossil deposits dating from as long as 700 million years ago, appear to have been flat, often segmented, but mostly solidbodied creatures. Among modern phyla the coelenterates, such as hydra, are forms with a single lumen; echinoderms (e.g. starfish) and vertebrates have both a digestive tube and a surrounding body cavity. It seems to have taken more than 100 million years after the appearance of the Vendian fauna for organisms to develop distinct body cavities. But when they arose in the early Cambrian, all the modern body plans burst onto the scene in short order. It has been speculated that the advent of polarized cells, and the straightforward physical consequences of this step, may have provided the basis for the rapid profusion of body types during the Cambrian explosion.

Interplay of ‘generic’ and ‘programmatic’ mechanisms of development The combined effects of the various physical properties that were generic to the earliest multicellular aggregates, considered as chemically excitable, viscoelastic ‘soft matter’, made virtually inevitable a profusion of multilayered, hollow, segmented forms early in the history of metazoan life. While the somatic organization of these ancient organisms resembled in many respects that of their modern counterparts, their developmental modes and mechanisms were profoundly different. In particular, many of these early organisms are likely to have been

polymorphic and morphologically interconvertible, by virtue of the conditional and interactive nature of the physical forces that moulded them. Only with the subsequent evolution of genetic redundancy and biochemical integration (in the course of which forms that originated by physical processes could be co-opted by ‘hardwired’ genetic circuitry) would organisms of the more familiar modern variety have emerged: entities in which bodily form is achieved with decreased participation of external physical forces and increased dependence on programmed genetic control.

The Epigenetics of Advanced Development Once basic body plans were established, selection for biochemical integration, promoting physiological homeostasis and developmental reliability, stabilized the relationship between genotype and phenotype. The role of genetic control in these more advanced developmental systems is undisputed, and its proximate workings in modern species represent the primary research focus of current developmental biology. But even in highly controlled forms of development the realization of morphology, particularly at the level of organogenesis, continues to depend on nonprogrammatic, epigenetic mechanisms. Among these are physicochemical, topological and biomechanical factors, as well as generic, stochastic and self-organizational properties of developing tissues, and the complex dynamics of interactions between these tissues. Although there is ample empirical evidence for the participation of these factors in individual ontogenies, their influence in setting trajectories of morphological evolution is only recently coming to be incorporated into the framework of evolutionary theory.

Nonprogrammed processes of development Genetically controlled development acts through a host of biomolecules by which cells regulate one another’s activities. But while in the very first steps of a developmental sequence the relationship between gene expression in a given cell and the resulting behaviour of itself and its neighbours can be direct and rather immediate, subsequent phases of development become characterized by increasing numbers of differentiated cell populations that produce molecular environments of ever greater variety and complexity. The composition of these local environments in turn influences cells in a broadening, combinatorial fashion, and the behaviour of individual cells becomes highly context-dependent. With intensifying complexity of the interactions among cells, genes and molecular environments, the developmental processes become increasingly removed from the direct and unique control by the


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genome. And unless specific mechanisms exist to oppose their actions, the generic properties of cells, cell products and tissues will always be of decisive importance in the organization of embryonic structures. Many of these contextual and generic determinants of development are rather distant from any programmatic influence by the genome. They include simple physical and geometric constraints exerted by growing cell masses, such as underlie the coiling of snail shells or the double spiral arrangement of sunflower florets. Others are the various forms of self-organization known to take place in cellular assemblies, which arise from differential adhesion, biochemical oscillations and reaction–diffusion coupling. These effects, highlighted above as sources of body plan diversity early in evolution, act at the tissue and organ level in the development of more evolved organisms, contributing to the formation of structures such as glands, respiratory organs, limbs and bristles. Instructions for general processes and even for very specific events in development can arise from the internal environment, such as maternal factors present in the egg, or the external environment (including the uterine environment in mammals), which can strongly influence development via its chemical composition, energy supply, temperature, humidity, gravity, mechanical stress, spatial confinement, illumination intensity and periodicity, etc., for all of which effects numerous empirical examples are known. Many events of early development, such as the initiation of the body axis or of epidermal differentiations, or events in late development, such as muscle individuation, innervation patterns or blood vessel growth depend on inductive interactions between tissue components, which are not deterministically programmed as to their site of occurrence. They merely require the meeting of a ‘sender’ and a ‘receiver’ tissue, often largely independent of time and location, and can depend on stochastic factors, such as cell number, size of the initial blastema, distance between competent tissues, etc. Finally, as documented in experiments perturbing embryo movement, the activity of the embryo itself contributes in many ways to the normal development of the size, shape, and arrangement of body components. This brief survey demonstrates that even advanced forms of genetically regulated ontogenies are under the influence of a host of nonprogrammed factors that contribute to the embryonic generation of form at least as significantly as the genetic component. The deployment of ‘genetic information’ is epigenetically controlled.

Rapid change and the origin of phenotypic innovation As a result of the prevalent role of epigenetic processes in both ancient and modern developmental systems, accounts of morphological evolution cannot be reduced to the 4

evolution of molecules and genes. The epigenetic component is responsible for many phenomena in morphological evolution not explained by classical neo-Darwinian theory, such as instances of rapid morphological change and the origin of new structural features at the subphylum level. Because gene change by mutation is relatively constant, the general expectation of the neo-Darwinian framework is that the rate of morphological change within a phylogenetic lineage should also remain rather constant. But, in strong contrast to this prediction, the empirical evidence from the fossil record documents numerous punctuated events. Molecular phylogenies also indicate that rapid changes of morphological structure and form have occurred in many recent species diversifications, but there are no indications that such episodes of rapid morphological change must be accompanied by accelerated genetic change. And analysis of inbred mouse strains and of developmental perturbation experiments also indicates that rapid, extensive changes of morphology are not necessarily linked to corresponding amounts of genetic change. This indicates that significant causal factors intervening between genome and form are missing from the standard model. It is the epigenetic character of developmental systems that supplies this critical explanatory level. In addition to providing insight into the relative independence of the tempo of morphological evolution from genomic changes, the epigenetic dimension of developmental systems can also help account for a second, almost enigmatic characteristic of morphological evolution, namely the appearance of new characters in a phylogenetic lineage. This puzzling problem is generally sidestepped by theoretical accounts of evolution, which usually concentrate on the variation and adaptation of given characters, but do not consider their origin and the mechanisms involved in their causation. However, the continuous addition of new morphological detail to the basic body plans is a general characteristic of evolving organisms and requires explanation. The generic, selforganizing, and conditional, interactive character of the epigenetic determinants discussed above provide a natural account for such morphological innovations. Genetic change, intraembryonic tissue interactions, and interaction of the organism with the external environment, can all lead to the crossing of thresholds in the equilibria of developmental interactions, and the reactive properties of the affected tissues can create kernels of new morphological structures. One example that has been studied in detail is the relation between the appearance of novel skeletal structures in the vertebrate embryo and alteration in tensile interactions among embryonic tissues (Mu¨ller and Streicher, 1989). The implication is that gradual evolutionary changes in the proportions of preexisting elements can abruptly generate such novelties as developmental thresh-



olds are crossed. These system-specific byproducts of the evolutionary modification of developmental processes appear as phenotypic innovations and are susceptible to becoming reinforced and stabilized in their realization by additional genetic change under standard Darwinian selection regimes. The process described represents a first step towards the establishment of new homologues in phylogenetic lineages.

The Origin of Morphological Homology Homology, the cross-taxic establishment of individualized and heritable anatomical building elements, embodies a key principle of biological organization. Homologues represent the natural units of evolutionary phenotype construction. Therefore any causal approach to the issue of morphological evolution must deal with the mechanisms underlying the origin of homologues. Here, again, epigenetics plays a central role. It is useful to distinguish three steps in the establishment of homology – namely the generation, the fixation and the autonomization of individual homologues – although clearly these processes will overlap. As discussed above, the first step, the generation of new elements, will often be a consequence of the epigenetic properties of developmental systems under modification. In contrast, the fixation of new homologues within existing body plans is the result of the progressive integration of phenotypic, developmental and genetic levels of interaction achieved by standard mechanisms of evolution. Phenotypic novelties initially brought about by generic means will increasingly rely on genetically controlled mechanisms for their ontogenetic realization. Redundancy in the genetic repertoire can be used for the control of new developmental interactions, resulting in an ever closer mapping between genotype and phenotype. Also, functional interdependencies that become established at the phenotype level will contribute to a further ‘locking in’ of new characters and will gain increasing organizational importance as more design and functional differentiation is added. Selection favours the genetic linkage of functionally coupled characters (Bu¨rger, 1986). But the evolution of homology does not stop at this point. Once new building elements have become integrated into the body design of a taxon, they can gain independence from the mechanisms responsible for their initial establishment. This is suggested by those cases in which different ontogenetic pathways are employed for the realization of the same structures in different species. Skeletogenesis in sea urchins, for example, involves the use of different progenitor compartments in direct developing species than it does in those with indirect development (Wray and Raff, 1989). The orbitosphenoid develops as membrane bone in amphisbaenids but as replacement bone in other verte-

brates (Bellairs and Gans, 1983). Meckel’s cartilage is induced by the endoderm in amphibians but by the ectoderm in higher vertebrates (Hall, 1983). It is well known that short germ band development in insects differs considerably from long germ band development, while the resulting structures are clearly homologous. And other studies show also that the molecular make-up and the genetic control of embryonic characters can change during evolution of a lineage. These examples demonstrate that the same phenotypic end point can be reached by alternative developmental modes and pathways. In other words, morphological homology persists while its molecular, genetic and developmental components become free to drift. But since the homologues themselves are maintained as construction units at the phenotypic level, they assume a role as independent organizers of body design. In this way phenotypic organization liberates itself from its mechanistic underpinnings and the evolution of morphological form becomes strongly determined by the specific set of homologues that a phylogenetic lineage has acquired.

References Bellairs AD and Gans C (1983) A reinterpretation of the amphisbaenian orbitosphenoid. Nature 302: 243–244. Bu¨rger R (1986) Constraints for the evolution of functionally coupled characters: A nonlinear analysis of a phenotypic model. Evolution 40: 182–193. Crick FHC (1970) Diffusion in embryogenesis. Nature 225: 420–422. Goldbeter A (1996) Biochemical Oscillations and Cellular Rhythms: The Molecular Bases of Periodic and Chaotic Behaviour. Cambridge, UK: Cambridge University Press. Hall BK (1983) Epigenetic control in development and evolution. In: Goodwin BC, Holder N and Wylie CG (eds) Development and Evolution, pp. 353-379. Cambridge, UK: Cambridge University Press. Mu¨ller GB and Streicher J (1989) Ontogeny of the syndesmosis tibiofibularis and the evolution of the bird hindlimb: A caenogenetic feature triggers phenotypic novelty. Anatomy and Embryology 179: 327–339. Newman SA (1993) Is segmentation generic? BioEssays 15: 277–283. Steinberg MS (1998) Goal-directedness in embryonic development. Integrative Biology 1: 49–59. Tsarfaty I, Rong S, Resau JH et al. (1994) The Met proto-oncogene: mesenchymal to epithelial cell conversion. Science 263: 98–101. Wray GA and Raff RA (1989) Evolutionary modification of cell lineage in the direct-developing sea urchin Heliocidaris erythrogramma. Developmental Biology 132: 458–470.

Further Reading Bonner JT (1996) Sixty Years of Biology: Essays on Evolution and Development. Princeton, NJ: Princeton University Press. Gould SJ and Lewontin RC (1979) The spandrels of San Marco and the panglossian paradigm. Proceedings of the Royal Society of London B 205: 581–598. Hall BK (1998) Evolutionary Developmental Biology, 2nd edn. London: Chapman and Hall. Kauffman SA (1993) The Origins of Order. New York: Oxford University Press.


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Larsen E (1992) Tissue strategies as developmental constraints: implications for animal evolution. Trends in Ecology and Evolution 7: 414–417. Mu¨ller GB (1990) Developmental mechanisms at the origin of morphological novelty: A side-effect hypothesis. In: Nitecki M (ed.) Evolutionary Innovations. Chicago, IL: University of Chicago Press. Mu¨ller GB and Newman SA (1999) Generation, integration, autonomy: Three steps in the evolution of homology. In: Homology (Novartis Foundation Symposium 222), pp. 65–79. Chichester: Wiley.

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Newman SA (1994) Generic physical mechanisms of tissue morphogenesis: A common basis for development and evolution. Journal of Evolutionary Biology 7: 467–488. Newman SA and Mu¨ller GB (1999) Epigenetic mechanisms of character origination. In: Wagner GP (ed.) The Character Concept in Evolutionary Biology. San Diego, CA: Academic Press, in press. Raff RA (1996) The Shape of Life: Genes, Development, and the Evolution of Animal Form. Chicago, IL: University of Chicago Press.
