The establishment, maintenance, and breaking of symmetry

The word symmetry is a derivative of symmetria and symmetros in Latin and Greek, meaning to have agreement in dimensions, proportion, and arrangement. The correct development of multicellular organisms depends on the establishment of symmetry both at the whole-body level and within individual tissues and organs. In biology, symmetry comes in many forms and is associated with beauty and functional necessity, which can have evolutionary or fitness advantages. Starfish are a classic example of radial symmetry, which can be halved in any plane to produce identical parts. In contrast, bilateral symmetry is defined by a single plane that divides an organism into two identical mirror-image halves. This is typical of the majority of animals on Earth, such as butterflies, for example. It would therefore be convenient to think of symmetry as a natural state for vertebrates and their embryos. However, there is also considerable evolutionary pressure to develop asymmetry in structures with high complexity, which drives variation, diversification, and adaptation. The breaking of symmetry is therefore also a fundamental feature of normal vertebrate development and is necessary to establish the anterior–posterior, dorsal–ventral, and left–right axes of the body plan. But how is symmetry established and maintained, and what are the evolutionary and developmental consequences of repeatedly breaking symmetry? Defining the mechanisms that establish, maintain, and break symmetry is fundamental to an improved understanding of development, evolution, and disease.

This Special Issue on “The Establishment, Maintenance and Breaking of Symmetry” contains a diverse selection of articles that explore some of the basic mechanisms that break symmetry during anterior–posterior axis formation and left–right patterning, including morphological structures such as the node and cilia, and the molecular pathways that drive asymmetric signaling, particularly the Nodal pathway. Asymmetry is a frequent feature of developmental disorders and the development and application of new tools for quantifying asymmetry can help reveal the genetic and environmental factors that drive the establishment, maintenance, and breaking of symmetry.

Breaking radial symmetry to establish anterior–posterior axis formation is a key developmental step in vertebrate gastrulation. The transient longitudinally oriented primitive streak is representative of the emerging anterior–posterior axis of birds and mammals. Pre-gastrulation pig embryos develop as a flat disc, the ancestral form of amniotes, and in this study,¹ Ploger and colleagues explore the expression and possible evolutionarily conserved function of Eomes, Tbx6, Wnt3, and Pkdcc in anterior–posterior axis formation. Similarities in expression patterns in pig embryos as compared to rabbit provide the first evidence for equivalence in the number of transient axial domains. Therefore, this study corroborates the opinion that axis formation, which occurs immediately prior to primitive streak formation, is conserved in mammals and may utilize a similar three-anchor point multistep mechanism to reliably establish axis formation and break symmetry during a key phase of development.

The process of gastrulation is quite variable across animal species, but it ultimately achieves the same end goal, which is the formation of the mesoderm germ layer and establishment of a triploblastic embryo. The node in avians and mammals is the equivalent of Spemann's organizer in amphibians, and together with the primitive streak is a transient structure that arises in the midline of the embryo during gastrulation. In this study, Harmoush et al.² examine the development of node architecture and the emergence of molecular organizer characteristics in the pig embryo. They examined the expression of select organizer genes prior to and during gastrulation in pig embryos. The node is a multilayered, dense columnar epithelium, with ventral mesenchymal cells that express Goosecoid, Chordin, and Brachyury in distinct domains defined as the gastrulation precursor domain, the presumptive node domain, and the mature node domain. Differences in size and morphological features such as epiblast epithelialization and notochord formation were noted. Whether the porcine node, with its distinct structure, exhibits inductive capacities or is functionally equivalent to Spemann's organizer in amphibians remains to be determined.

The Nodal and Lefty genes are members of the transforming growth factor-beta (TGFβ) superfamily. They regulate the expression of intercellular signaling molecules and are crucial for left–right symmetry breaking. However, they also exhibit distinctive features within their gene repertoires. The extent of conservation of Nodal and Lefty genes between different animal lineages is controversial, but phylogenetic reevaluation and synthetic interpretation by Kuraku³ now provide a revised nomenclature for these genes covering the entirety of vertebrate diversity. Scanning genome-wide sequences revealed unusual patterns of gene repertoire evolution with reciprocal gene loss, gene conversion, and positional intervention of other genes. This work exposes hidden paralogy between Nodal1 and Nodal2 genes resulting from differential gene loss in amniotes, and the tandem cluster of Lefty1 and Lefty2 genes, which was thought to be confined to mammals, was found in sharks and rays, with an unexpected phylogenetic pattern. This article therefore provides a comprehensive review of the origins of these vertebrate gene repertoires and proposes a revised nomenclature based on vertebrate genome evolution.

Epidermal Growth Factor -like motif and a novel sequence, first identified in mouse Cripto, frog FRL-1, and mouse Cryptic/Cfc1 (EGF-CFC) proteins are best known for their roles as co-receptors for Nodal signaling during gastrulation, as well as anterior–posterior, dorsal–ventral, and left–right patterning. Vertebrate species with multiple family members exhibit evidence of functional specialization. Shylo and Trainor⁴ describe the evolutionary history of the EGF-CFC family of proteins in deuterostomes, with a focus on vertebrates. Taking advantage of the relatively new abundance of high-quality sequenced and annotated deuterostome genomes, the authors trace the evolutionary history of the EGF-CFC family of proteins in deuterostomes from a single gene through their expansion in tetrapods, then specialization, gene loss, and translocation in eutherian mammals. EGF-CFC proteins have been historically reported to have little sequence conservation between species, beyond the CFC-EGF domain, which remains relatively well conserved. Mouse Cripto and CFC1, zebrafish Tdgf1, and each Xenopus EGF-CFC gene (Tdgf1, Tdgf1.2, and Cripto.3) are all descendants of the ancestral deuterostome Tdgf1. Subsequent to EGF-CFC family expansion in tetrapods, Tdgf1B (Xenopus Tdgf1.2) appears to have acquired specialization in the left–right patterning cascade, and then after its translocation in eutherians to a different chromosomal location, CFC1 maintained that specialization.

Vertebrate left–right symmetry breaking is preceded by the formation of the left–right organizer. In amphibians, the gastrocoel roof plate, which is covered by ciliated epithelium of mesodermal fate, is regarded as the left–right organizer. The gastrocoel roof plate is equivalent to the ciliated posterior notochord in mammals and is designated as the ventral node. Petri et al. performed a detailed analysis of the spatial and temporal expression of important genetic markers in concert with the morphology of the emerging gastrocoel roof plate.⁵ The gastrocoel roof plate can be subdivided into a medial area, which generates leftward flow by rotating monocilia, and lateral Nodal1 expressing areas, which sense the flow. After symmetry breaking, medial cells are incorporated into a deep layer where they contribute to the axial mesoderm, whereas lateral domains become part of the somitic mesoderm. Overall, this work prompts new questions about the evolution and divergence of left–right symmetry breaking mechanisms in vertebrates.

In mouse, the left–right organizer is called the node. It is composed of two types of ciliated cells: pit cells at the center that possess motile cilia that generate the leftward nodal flow, which is responsible for establishing left–right determination; and crown cells, which have immotile (primary) cilia that occupy the periphery of the node. Leftward flow mechanically activates the Pkd2 channel on immotile cilia and increases the frequency of calcium transients and asymmetrical bending of the cilia on the left side. Katoh et al.⁶ evaluated the effects of key signaling molecules, and most notably Bone Morphogenetic Protein 4 (BMP4), which is expressed in the lateral plate mesoderm, on the asymmetric distribution of Pkd2. Optical tweezer manipulation of nodal immotile cilia revealed that excess BMP4 perturbs the mechanosensing ability of the cilia. BMP4 influences the asymmetric distribution of Pkd2 in nodal immotile cilia, which affects the ability of these cilia to sense the bending direction for left–right determination. This study illustrates the importance of asymmetric protein distribution in cilia and their function.

The craniofacial complex is derived from lateral outgrowths termed facial prominences and pharyngeal arches, which are composed of neural crest cells, surface ectoderm, and mesoderm. These primordia grow and fuse to form specific structures in the head and face such as the jaw, which are bilaterally symmetrical. Not only is such symmetry actively generated and maintained, but asymmetry is buffered against. Perturbation of this multifaceted morphogenetic process, however, can result in facial asymmetry or premature fusion of primordia, or prevent apposition and fusion of primordia, leading in each case to craniofacial anomalies. Receptor tyrosine kinase signaling is a critical driver of the localized cellular proliferation, apoptosis, migration, and differentiation that underpins proper craniofacial morphogenesis. In this study, Hanne et al.⁷ inhibited MEK1/2, PI3K, and PLCγ pathways, which are major downstream effectors of receptor tyrosine kinase signaling, to investigate their roles in regulating these specific cellular activities during craniofacial development. Receptor tyrosine kinase signaling is ubiquitous and robust to developmental perturbation through redundant signaling systems. However, precise regulation of receptor tyrosine kinase signaling is required to control regional cellular activities such as proliferation and growth, ensuring proper bilateral symmetry in craniofacial development and function.

Asymmetry is not only a key feature during normal development, but also a characteristic of developmental disorders such as schizophrenia, autism spectrum disorder, fetal alcohol syndrome, and structural birth defects, such as orofacial clefting and microtia. Asymmetry can therefore be used as a readout of genetic variation and environmental perturbation. Rolfe et al.⁸ introduce a tool that can be run within the 3D Slicer application to quantify asymmetry in 3D images. They then apply their approach to explore the role of genes contributing to abnormal craniofacial asymmetry by deep phenotyping of 3D fetal micro-computed tomography images from mutant mice acquired through the Knockout Mouse Phenotyping Program. These and other large-scale imaging datasets can provide the critical sample sizes necessary to detect and quantify differences in morphology. Together with the development and application of tools for precisely quantifying asymmetry, this can facilitate a better understanding of the genetic basis of asymmetry and its relationship to disease susceptibility. Ultimately, these resources and tools will help unravel the complex genetic and environmental factors and their interactions that increase risk in the etiology and pathogenesis of developmental disorders.