Inherency of Form and Function in Animal Development and Evolution
Lateral inhibition mediated by the Notch pathway is well suited to generating fine-grained salt-and-pepper-like patterns in which adjacent cells are of different types. Organogenesis in complex animals, however, depends on large-scale coordination of patterns of cell differentiation in which patches and domains of cells of a given type—tissues—associate in specific geometric arrangements with other such domains in the architecture of body layers, appendages, and primordia arising from the morphogenetic processes described above. Morphogen gradients, mentioned above, can mediate long-range control of differentiation, but in order to effect accurate pattern formation, the cells in the targeted regions need to be in similar states of receptivity. This might appear to be a property that is not intrinsic to cells in developing tissue primordia and must be a product of adaptive selection. But, it can in fact arise spontaneously from mechanisms and ingredients that have evolved for other reasons.
A main target gene of the CSL transcription factors at the end of the Notch signal transduction pathway is Hes1 (called hairy1 in Drosophila). Hes1, itself a transcriptional regulator, has the unusual property of regulating its own gene expression through a negative feedback mechanism. Due to this feedback control, the level of Hes1 protein in expressing cells will tend to undergo temporal oscillation (Lewis, 2003). Then, through a well-described physical phenomenon of synchronization of weakly coupled oscillators (Garcia-Ojalvo et al., 2004), the cells in a spatially extended domain of tissue will find themselves in identical though periodically changing states of Hes1 concentration and of any other biochemical factor Hes1 controls. This poises them for concerted response to an external regulatory factor such as a morphogen. The Notch pathway and its downstream effectors, therefore, not only enable cells to directly influence the fates of their nearest neighbors through lateral inhibition, but facilitate the emergence of a “morphogenetic field” (Gilbert et al., 1996; Bhat et al., 2019). This phenomenon was described by early embryologists as “a system of order such that the positions taken up by unstable entities in one part of the system bear a definite relation to the position taken up by other unstable entities in other parts of the system” (Needham, 1937, p. 71).
The cell state-coordinating effect of synchronized oscillations of Hes1 expression or potentially other factors with similar feedback dynamics has a propensity (not invariably realized) of producing spatially periodic tissue structures. Hes1 oscillations are synchronized, for example, in the lateral plate mesoderm of vertebrate embryos, so that cells in any band of tissue along the cranial-caudal axis have approximately the same (periodically varying) Hes1 concentration (Özbudak and Lewis, 2008). The cells will aggregate, due to upregulation of adhesive molecules, when a certain Hes1 level is reached, but only when they are not inhibited from doing so by a graded suppressive activity, a morphogen, that emanates from the tail tip. As the embryo elongates, successive bands of tissue become distant enough from the caudal end to escape from the morphogen’s effect. When the Hes1 oscillation brings the factor’s concentration into the adhesion-inducing range in the newly competent cells (uniformly, because of synchrony), a mesenchymal-epithelial transformation takes place, and discrete blocks of tissue, “somites,” form to either side of the central axis (Hubaud and Pourquie, 2014). This process continues as long as the embryo elongates, generating a species-characteristic number of somites, which depends on both the rate of elongation and the period of the oscillator. While the somites give rise to various spatially periodic (the vertebrae and the ribs) and nonperiodic (the dermis of the back skin, and the muscles of the back, body wall, and limbs) structures, the manner in which they are generated suggests that the emergence of tandem tissue blocks is the side effect of various inherent processes (feedback regulation, morphogen action) that only incidentally, under certain mutually tuned conditions, lead to a repetitive spatial pattern.
In a similar fashion, Hes1 oscillations across the digital plate in the embryonic avian limb become synchronized during skeletal pattern formation. This enables spatial cues for skeletogenesis (see below) to act in a coordinate fashion over larger distances across the cellular field than the natural wavelength of their generating mechanisms (Bhat et al., 2019).
Morphogens do not only function in the form of simple gradients to induce patterns of cell differentiation. By interacting with biosynthetic processes (including gene expression) in target cells and tissues, they can assume more elaborate geometries, which lead to more complex cell arrangements. Alan Turing, in a paper titled “The chemical basis of morphogenesis,” showed that a balance of positive and negative feedbacks in an open chemical system, when coupled with differences in the rates of diffusion of the key reactive molecules, could lead (contrary to the expectation that diffusion evens things out) to stable, nonuniform concentration patterns (Turing, 1952). These are referred to as “reaction-diffusion” mechanisms, and in developmental biology, “Turing-type” systems, but when living tissues are concerned neither “reaction” (actually cell-based production of, and response to, molecular factors) nor “diffusion” (or more generally, transport of released molecules through and between cells, with the latter’s active participation), are typically like those seen in purely chemical systems. Moreover, the core processes may differ substantially from Turing’s, lacking some of the original features, e.g., the difference in diffusivities of the chemical species involved, and containing others, e.g., chemotaxis and advection (see, for example, Glimm et al., 2014; Madzvamuse et al., 2015; Nesterenko et al., 2017).
Turing-type mechanisms pattern cell types in a variety of embryonic systems, including spatially nonperiodic ones such as the generation of left-right asymmetry (Muller et al., 2012) and the formation of individual tooth cusps (Salazar-Ciudad, 2012). But, it is in the production of repetitive or quasi-repetitive structures such as hairs and feather germs (Glover et al., 2017; Painter et al., 2018), and pigment patterns in skin (Haupaix et al., 2018), that this category of self-organizing process has found its broadest applications. The paired appendages – fins or limbs – of the jawed vertebrates, for example, are characterized by arrangements of cartilaginous and/or bony elements that appear to have arisen evolutionarily by and continue, to some extent, to be mediated by such mechanisms. In cartilaginous fishes such as sharks, skates, and rays, the fin skeleton consists of one or more cartilage rods or plates to which are appended as many as several dozen parallel, jointed cartilage rods. In ray-finned fishes (carp, zebrafish, and so forth), the endoskeleton is made of plates, rods, and nodules of cartilage and bone. Finally, in the lobe-finned fish like the coelacanth, and tetrapods, such as amphibians, birds, and mammals, the cartilaginous or bony skeleton is comprised of increasing numbers of parallel elements along the proximodistal axis originating at the body wall (Newman et al., 2018).
Evidence from experimental embryology and molecular genetics, phylogenomics, and mathematical and computational simulations has provided plausible scenarios for transitions, often abrupt, between these patterns over the course of evolution of the paired appendages resulting from subtle changes in transcription factor-cis-regulatory element binding within conserved GRNs, and protein-protein interactions at the cell surface. In these cases, and in others which have been explored in detail, active and excitable mechanisms of cellular differentiation and pattern formation, in coordination with more passive morphogenetic mechanisms of the liquid- and liquid crystalline-tissue states, each utilizing genes, molecules, and regulatory motifs particular to metazoan life, lead to the formation of recognizably animal-type tissues and organs with the capacity to perform functions carried over from ancestral cells.
This review has described how three main properties of animal bodies and organs, their forms, their differentiated cell types, and the patterned arrangement of cells of various types, are determined by inherent properties of aggregates of metazoan cells. These inherent properties are not all of the same type. The only such properties that were actually latent in directly ancestral unicellular organisms are those manifested as the specialized functions of cell types. As indicated above, unicellular holozoans are variously capable of adhering to one another, absorbing molecules and ingesting particulate nutrients, exhibiting contractility and electrical excitability, surrounding themselves with semisolid or solid matrices, binding oxygen, neutralizing toxins, and excreting waste products. All of these functions appear in exaggerated form in one or more of the 200–300 animal cell types. Functions inherent to the ancestors of other multicellular lineages, such as the capture of light and the storage of its energy in sugars in vascular plants, appear in differentiated cells of those organisms, but not the animals.
As noted above, the recruitment of inherent cell functions for cell differentiation sometimes employed transcription factors which controlled the relevant function-related genes in the unicellular ancestor. Most often, however, the differentiation and other developmental TFs are not found outside the metazoans (Grau-Bové et al., 2017). In all cases, however, the TFs involved in cell differentiation became incorporated into hierarchies, based on the novel and unique gene regulatory apparatus of Metazoa that distinguished their roles in establishing cell types from those in maintaining them, and set both of these apart from regulation of gene expression in nonspecialized “housekeeping” activities.
The relevant organizational properties for morphogenesis and pattern formation are those characteristic of mesoscopic materials (e.g., liquid- and liquid-crystalline tissues) or extended excitable media (e.g., “diffusion” and “reaction,” synchronization of oscillations). While the respective physical effects are therefore generic to such materials, the concept of inherency was only applicable once the materials came into existence as multicellular masses. It is notable that gene innovations that are specific, or now uniquely confined, to Metazoa were required for such materials to be constituted and thus for the inherent properties of their physical state to be realized.
The fact that body and organ forms, cell and tissue functions, and cellular patterns are inherent to animal systems has major consequences for understanding evolution (Newman, 2017). (See Webster and Goodwin, 1996 and Amundson, 2005 for accounts of the structuralist tradition in evolutionary theory, in which inherency is an important theme.) Inherency clearly does much of the work attributed in the standard model to trial-and-error-based natural selection. But inherency of form and function does not mean simultaneous emergence of all possibilities. The main animal types do not appear in the fossil record prior to strata deposited tens of millions of years after the first signs of metazoan-type organisms, more than 600 million years ago. With the exception of placozoans and ctenophores, which, respectively, implement the developmental gene-regulatory and liquid-tissue constituting activities of enhancers and classical cadherins differently, the capability to mobilize pre-existing holozoan cell functions in morphologically varied, spatially patterned assemblages was present from the start and carried forward in all subsequent evolution of this group. Additional complexity was achieved mainly by the addition of new genes specifying ECM components.
Finally, the remarkable phenomenon of electrical integration of multicellular patterns must be mentioned. Although thus far not demonstrated to be a primary form-generating or patterning mechanism like differential adhesion, morphogen gradients, gene expression oscillation, and reaction-diffusion coupling, bioelectric establishment of voltage gradients across tissues serves to reinforce developmental patterns and restore them if perturbed during embryogenesis or wounding (McLaughlin and Levin, 2018; Pietak and Levin, 2018). Voltage-gated channels with single-cell functions are inferred to have existed in unicellular holozoan ancestors of the animals (Cai, 2012), but their developmental role in the multicellular context is entirely different from any function they evolved to perform in individual cells. Along with canalizing evolution that serves to stabilize the phenotype of an evolutionary lineage against mutation and developmental noise once it has become ecologically established, the dynamically responsive encoding of morphology by bioelectricity is a key factor in organismal autonomy (Arnellos et al., 2014; Moreno and Mossio, 2015).
Major steps in animal evolution not only occurred by such repurposing of ancestral genes (as we also saw in the case of the grainyhead transcription factor), but also involved in the appearance, often without precedent, of novel genes whose products set into play forces and effects, which though having predictable outcomes, were unavailable up to the points at which they appeared (Table 1).
Natural selection can act as a sieve for organisms with novel combinations of inherent characters, or characters that become inherent to a lineage after genetic augmentation, but it is not responsible for producing those traits. The characters’ origination, whatever the source of the associated genes, can often only be understood on the basis of physico-genetic effects specific to the multicellular context. Characters produced in this fashion are constrained in their nature and may therefore appear abruptly (McGhee, 2011). If they enable organisms to survive in new ways in existing ecological niches, or to occupy new niches (Laland et al., 2017), their roles in enhancing fitness will be “after-the-fact” (Gould and Lewontin, 1979; Müller, 1990; West-Eberhard, 2003) and thus do not require elaborate or farfetched adaptationist narratives to account for their existence.
The author confirms being the sole contributor of this work and has approved it for publication.
The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
I thank Johannes Jaeger and two anonymous reviewers for their close reading, detection of errors in, and constructive comments on, the original text, and suggestions of additional relevant material.