*Review* **Atypical Brain Asymmetry in Human Situs Inversus: Gut Feeling or Real Evidence?**

**Guy Vingerhoets \* , Robin Gerrits and Helena Verhelst**

Department of Experimental Psychology, Ghent University, 9000 Ghent, Belgium; robin.gerrits@ugent.be (R.G.); helena.verhelst@ugent.be (H.V.)

**\*** Correspondence: guy.vingerhoets@ugent.be

**Abstract:** The alignment of visceral and brain asymmetry observed in some vertebrate species raises the question of whether this association also exists in humans. While the visceral and brain systems may have developed asymmetry for different reasons, basic visceral left–right differentiation mechanisms could have been duplicated to establish brain asymmetry. We describe the main phenotypical anomalies and the general mechanism of left–right differentiation of vertebrate visceral and brain laterality. Next, we systematically review the available human studies that explored the prevalence of atypical behavioral and brain asymmetry in visceral situs anomalies, which almost exclusively involved participants with the mirrored visceral organization (situs inversus). The data show no direct link between human visceral and brain functional laterality as most participants with situs inversus show the typical population bias for handedness and brain functional asymmetry, although an increased prevalence of functional crowding may be present. At the same time, several independent studies present evidence for a possible relation between situs inversus and the gross morphological asymmetry of the brain torque with potential differences between subtypes of situs inversus with ciliary and non-ciliary etiologies.

**Keywords:** situs inversus; heterotaxy; brain asymmetry; visceral asymmetry; vertebrate asymmetry; human laterality; left-right differentiation; brain torque; ciliopathy

#### **A glossary of terms is available at the end of this paper**

#### **1. Introduction**

Vertebrates' visceral and central nervous systems demonstrate a strikingly asymmetric organization with a strong population bias toward a prototypical left–right configuration [1,2]. As both systems serve fundamentally different biological functions, it seems plausible to assume that the reasons behind their asymmetry may be entirely different and that their left–right differentiation evolved independently. While this may be true, it does not preclude the possibility that basic mechanisms for establishing left–right differentiation of the viscera have been reused to establish central nervous system laterality and that there may be a link between both manifestations of asymmetry. The strong population bias in visceral and brain asymmetry makes it difficult to determine whether they develop independently or related. Research turned to atypical conditions of visceral laterality to investigate possible relationships. Animal studies showed that some species like newts and zebrafish appear to align their brain and visceral asymmetry, mediated by *nodal*related events [3–5]. In the frequent-situs-inversus (fsi) line of zebrafish, visceral reversal is accompanied by neuroanatomical reversals in the diencephalon, particularly epithalamic nuclei, which are believed to be involved in the functional lateralization of the vertebrate central nervous system [6]. In line with this claim, diencephalic reversals of fsi zebrafish correlate with the reversal of some (but not all) lateralized behavioral responses [7]. Do we anticipate a similar association in humans?

We will approach this outstanding question by describing the phenotypes and development of left–right asymmetry of the visceral system and the central nervous system

**Citation:** Vingerhoets, G.; Gerrits, R.; Verhelst, H. Atypical Brain Asymmetry in Human Situs Inversus: Gut Feeling or Real Evidence?. *Symmetry* **2021**, *13*, 695. https:// doi.org/10.3390/sym13040695

Academic Editor: Sebastian Ocklenburg

Received: 24 February 2021 Accepted: 14 April 2021 Published: 16 April 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

and discuss the possible links between their mechanisms of left–right differentiation. Ultimately, a valid test for the hypothesis of an association between human visceral and neural asymmetry is to investigate the prevalence of atypical brain asymmetry in participants with visceral situs anomalies. In a systematic review, we discuss the studies that provide empirical evidence on behavioral, brain functional, and brain structural asymmetries in participants with situs anomalies. However, first, we will briefly explain the relevance of studies on asymmetry for the evolution of development.

Fluctuating asymmetry, directional asymmetry, and antisymmetry constitute three observable types of asymmetry within a population. Fluctuating asymmetry is the amount of deviation from perfect bilateral symmetry, and it manifests as small differences between the left and the right sides due to random errors in individual development. Fluctuating asymmetry is caused by genetic or environmental stress and is taken to measure developmental instability reflecting the level of stress in populations or of individual quality [8]. Directional asymmetry refers to the phenomenon that most individuals in a population are asymmetrical in the same direction, whereas in antisymmetry, dextral and sinistral forms are equally present within a species [9]. The typical asymmetrical position of the internal organs in vertebrates is an example of directional asymmetry, and the equal number of male fiddler crabs with a larger left or right claw is the prototypical example of antisymmetry. The latter two types of asymmetry have been proposed as informative traits to investigate evolution mechanisms as they are easy to define, easy to compare, and have evolved multiple times independently [9]. Differences in the heritability of antisymmetry (absent) and directional symmetry (present) contribute to understanding the evolutionary origin of novel forms, and it has been posited that directional asymmetry appears to have evolved through genetic assimilation (phenotype precedes genotype) almost as frequently as through conventional mutation-mode (genotype precedes phenotype) [9]. Comparing asymmetry patterns across species is relevant to investigate the evolutionary history of gene-expression patterns and anatomical asymmetries. The nodal signaling cascade, which takes a central place in vertebrate asymmetry, provides an important example of cascade capture and trait canalization [9]. In fact, a comparison of the key nodal cascade genes in lower chordates and vertebrates surprisingly suggests that the ancestral target of the nodal cascade might have been brain asymmetry [9].

#### **2. Left–Right Asymmetry of the Visceral System**

#### *2.1. Phenotypes of Situs Viscerum*

Like all vertebrates, humans establish left–right asymmetry of the thoracic and abdominal organ position during embryogenesis [1,10]. The position (*situs*, Latin) of the internal organs (*viscera*, Latin) in the human body shows a strong population bias toward an asymmetric organization with the heart's apex and aorta, bi-lobed lung, stomach and spleen on the left side of the body midline, and the heart's vena cava, most of the liver and the tri-lobed lung on the right side [11]. This typical configuration is called situs *solitus* (from Latin, meaning habitual), presents in about 99.99% of the human population and is taken to reflect optimal packing and transfer of body fluids [11]. Anomalies of this arrangement span a wide range of laterality defects whose classification remains without general consensus, thus hampering pathological, genetic, and epidemiological research [12,13]. As etiological and morphological boundaries between atypical manifestations of visceral situs remain to be settled, there is general agreement on the main two phenotypic subgroups of situs anomalies; the complete or partial reversal of the typical condition termed situs *inversus* (from Latin, meaning inverted), and the mirroring of either the typical left or right visceral configuration, called *heterotaxy* (from Greek *heteros*: other, different and *taxis*: arrangement) (Figure 1). As a rule, situs inversus and heterotaxy occur in different families, but occasionally they present in the same (often consanguineous) family [14,15]. Epidemiological studies estimate the prevalence of human visceral laterality defects between 1/5000 and 1/11,000 live births [12,16,17].

**Figure 1.** Anatomical dispositions of the viscera in the different types of "situs" (radiological convention). Reprinted with kind permission by Dr. Francisco Barqueros Escuer, https://dx.doi.org/10.26044/ecr2019/C-2735 (accessed on 1 October 2020).

#### *2.2. Situs Inversus*

Complete reversal of the standard visceral arrangement with the heart now in a right-sided position (*dextrocardia*) is referred to as *situs inversus totalis*. Prevalence reports vary widely and have been estimated between 1/6000–1/33,000 live births [12,17,18]. The condition itself is not associated with adverse medical complications as complete mirroring through the midsagittal plane of organs, blood vessels, nerves, and lymphatics do not interfere with their morphology nor positional relationships [11,19]. People with situs inversus totalis can live perfectly healthy lives, and medical problems may arise only in case of organ transplantation/donation or atypical symptom lateralization (for example, in appendicitis). Because of its limited clinical repercussions, situs inversus totalis is believed to be underdiagnosed. Nevertheless, structural malformations, such as congenital heart disease, may occur more frequently in situs inversus than in situs solitus [20,21]. In rare cases (1/2,000,000), situs inversus is not complete, and the heart is in its usual position (*levocardia*), while the other organs are in reversed position. Isolated levocardia is often associated with severe cardiovascular malformations because of the heart's unusual position compared to the other organs and their connections [20]. In about a quarter of cases, situs inversus occurs as part of a congenital syndrome in which medical complications are more prominent [11]. One of these syndromes, primary ciliary dyskinesia, has elucidated the importance of tiny hair-like organelles (cilia) in the ontogenesis of visceral asymmetry and will be discussed in more detail below.

#### *2.3. Heterotaxy*

An entirely different type of situs anomaly is heterotaxy, also referred to as situs *ambiguus*, as the defect presents as a complete loss of left–right laterality in the arrangement of the visceral organs along the superior–inferior axis. In contrast to situs inversus, heterotaxy

syndrome alters the structure of visceral organs, particularly the heart, including the attachment of the large blood vessels, with the major morbidity and mortality resulting from complex cardiovascular malformations [13,16,22]. Prevalence figures for heterotaxy are estimated at 1/8000–1/12,000 live births [12,16,17]. Although classic heterotaxy accounts for only 3% of all congenital heart defects, gene mutations causing heterotaxy are also known to result in isolated cardiovascular malformations with no other visceral abnormalities, suggesting that the real prevalence of genetic heterotaxy is probably higher [19,23]. Two general types of heterotaxy, called isomerism, are described, although their exact morphology and its resulting abnormalities vary from patient to patient [11,19]. In left isomerism, morphologically left structures present on both sides of the body in the same individual. In this case, atrial cavities are morphologically left, both lungs will be bi-lobar with long main bronchial branches, the spleen is present but consists of multiple small and poorly functioning parts (polysplenia). In right isomerism, the right-sided visceral configuration is copy-mirrored to the left resulting in morphologically right atrial cavities, two tri-lobar lungs with short main bronchi, and an absent spleen (asplenia). In both conditions, the morphologically altered liver lies across the midline of the body, and intestinal malrotation is a typical feature, as well as cardiac malformations, the latter being more severe and sometimes life-threatening in right isomerism.

#### *2.4. Cause of Visceral Situs Anomalies*

Situs viscerum anomalies are congenital conditions due to heterogeneous genetic mutations that impact left–right patterning in early embryogenesis [19]. Genes involved in left–right axis development have emerged from animal studies and reveal a complex genetic cascade of left–right differentiation prior to the appearance of morphological asymmetry [14]. Most situs anomalies occur due to sporadic mutations, and many different genetic factors or genes cause the condition among different people or families [24]. Environmental and stochastic influences may also play a role as in a substantial number of cases, no clear monogenetic basis for their condition can be found [25]. In some families, situs viscerum anomalies present with an autosomal dominant, autosomal recessive (most commonly), or even X-linked pattern of inheritance [11,19]. Situs anomalies may arise as a variable manifestation of a syndrome encompassing a broader spectrum of defects [11]. Situs inversus, for example, sometimes occurs in cystic renal disease, Bardet-Biedl syndrome, and retinitis pigmentosa [24]. The best-known example of syndromal situs inversus, however, is when situs inversus arises as a symptom of primary ciliary dyskinesia (PCD), accounting for about 20 to 25% of its cases [19,21,26]. Primary ciliary dyskinesia is a causally heterogeneous group of autosomal recessive disorders characterized by a defect in the motility of small hair-like organelles (cilia) that protrude from the cell surface into extracellular space and perform various transport-related functions in the human body [27,28]. Ciliary motility is important for moving fluids and particles over epithelial surfaces, and cilia play crucial roles in various signal transduction pathways. Motile ciliogenesis requires a complex genetic program, and mutations of involved genes have been associated with ciliopathies, including primary ciliary dyskinesia (*DNAH5*, *DNAH11*, *DNAI1*, . . . ) [26,28–30]. Ciliopathies give rise to a complex spectrum of disease and developmental mutant phenotypes that can be organ-specific or have broadly pleiotropic effects [31]. The diagnosis of primary ciliary dyskinesia is commonly based on electron microscopy showing abnormalities in structure and function of dynein arms or outright absence of cilia [26]. Affected individuals (1/10,000 to 1/20,000 live births [30,32]) have chronic upper respiratory tract (sinusitis) and lower respiratory tract (bronchiectasis) infections as well as chronic ear infections (otitis media) due to defective mucociliary clearance [26,29,33]. Reduced male fertility caused by decreased sperm motility, variable female infertility, and decreased sense of smell can also be part of the spectrum. About half of the patients with primary ciliary dyskinesia and associated sinusitis and bronchiectasis also have situs inversus (a triad of symptoms known as Kartagener syndrome [34]), while the other half is situs solitus [29]. Given the specificity of the ciliary mutation causing visceral

inversion versus those causing respiratory problems, most but not all subgroups of the PCD syndrome will affect the genetic cascade induced by ciliary motion at the embryonic node (see below). Hence, the incidence of situs inversus in primary ciliary dyskinesia is estimated slightly less than the often reported 50%, and the Kartagener triad is expected in 1/22,000 live births [32]. Cardiac malformations suggestive of heterotaxy are found in 6–12% of individuals with primary ciliary dyskinesia [22,35], but it is generally believed that the condition is associated with a (near) randomization of left–right directionality rather than a loss of left–right specification [14]. The occurrence of a monozygotic twin pair with primary ciliary dyskinesia and with discordant visceral situs underlines the arbitrary nature of situs directionality in this condition [36].

#### **3. Left–Right Visceral Development**

#### *3.1. Motile Cilia at the Primitive Node*

The vertebrate left–right axis is established after developing its dorsal–ventral and anterior–posterior axes, and it is crucial for the correct positioning and morphogenesis of the internal organs [1,10]. The formation of the left–right axis involves several steps that have been investigated in several model organisms, such as the frog, zebrafish, chick, pig, and mouse (for a more detailed account, see [1,10,31,37–39]). While some genetic mechanisms are shared between vertebrates (like the expression of *nodal*, *lefty1*, *lefty2* and *pitx2*), other steps of the process seem to have diverged in evolution [10]. In fact, variation in the nodal cascade among vertebrates was said to resemble an hourglass, a conserved core set of genes listed above, with divergent genetic elements upstream and downstream that largely outnumber the shared core [9]. In most model organisms, symmetry breaking is established at the primitive node, a short-lived embryonic cavity filled with extracellular fluid that forms at the anterior tip of the primitive streak, a line of cells that establishes bilateral symmetry in the embryo, marks its future posterior side, and signals the beginning of gastrulation. Gastrulation is an important period in embryogenesis, which essentially consists of the differentiation of cells into an ectoderm, mesoderm, and endoderm layer. The left–right organizer or primitive node develops about 17 days postovulatory. The formation of the node coincides with the formation of motile cilia whose rotation produces a coordinated and unidirectional flow of the extracellular fluid that will induce symmetry breaking during gastrulation. It is important to point out that earlier asymmetries in the localization of some molecules have been established in some species and it has been claimed that asymmetries might exist perhaps as early as fertilization [37,40]. It is also important to note that not all species have fluid producing nodal cilia (absent in the chick and pig) yet show similarly strong population asymmetries of the viscera, which suggests that alternative cilia-independent symmetry breaking mechanisms at the node exist or that the cilia function as transmitters or amplifiers, but not initiators, of the asymmetrization [40]. In any case, in species with nodal cilia, such as the mouse, fish, and frog, (experimental) disruption of cilia functioning results in situs anomalies [41,42]. Reversal of flow in wildtype embryos results in L–R inversion, and introducing a leftward flow in mutants with ciliopathy restores typical L–R asymmetry [42,43]. While these experimental manipulations of ciliary flow are, of course, not possible in human embryos, the Kartagener syndrome clearly establishes humans as a species in which ciliary malfunction impacts visceral asymmetry. It may seem strange that a lack or impaired nodal flow caused by dysfunctional or absent cilia would result in L–R inversion instead of randomization, but models have been proposed to explain this [44].

#### *3.2. Propagation of the Signal to the Lateral Plate Mesoderm and Organ Primordia*

Due to their tilt and chiral nature, cilia that arise from nodal cells at the center of the nodal pit produce a clockwise (from tip to base) rotational motion that creates a leftward "nodal flow" towards the left periphery of the node [45] (Figure 2). Fluid flow is sensed by mechanosensory and/or chemosensory cilia in peripherally-located crown cells at the lateral ends of the pit [42]. These events cause intracellular Ca2+ levels to

increase on the left side of the node, which results in asymmetries in gene expression and the establishment of a L–R axis [31]. The resulting asymmetric gene expression is then propagated to the lateral plate mesoderm—sheets of embryonic tissue at the peripheral left and right side of the embryo that will form the body wall and circulatory system—where a cascade of asymmetric left-sided gene expression is established (*nodal*, *lefty2*, *pitx2*). Several mechanisms have been proposed to explain the propagation of signaling from the node to the lateral plate mesoderm either directly by diffusion of *nodal* or by a cascade of signaling events via sonic hedgehog (*shh*) or bone morphogenetic protein (*bms*) that asymmetrically affect nodal expression [1]. In any case, the expression of *nodal* and the *lefty* genes (nodal antagonists) is transient and exclusively on the left side [1]. Finally, this asymmetric signaling is propagated from the lateral plate mesoderm to organ primordia for proper morphogenesis of the viscera to occur (*pitx2*). It is proposed that *nodal* acts as a determinant for leftness because cells that receive nodal signals will adopt left-side morphology, and those that lack nodal signals will adopt right-side morphology. In mutations in which *nodal* is bilaterally expressed in the lateral plate mesoderm, embryos will develop left isomerism, and in those that lack nodal signal on either side, embryos will develop right isomerism [1]. While heterotaxy may result from deficits in any of the above steps, they more often occur at one of the later stages. Situs inversus, on the other hand, is believed to originate from a more initial deficit in nodal flow caused by defectively operating cilia when the total direction of left–right asymmetry is determined. Animal models identified over 100 genes involved in left–right patterning, and more are to come [24]. Their mutations, in combination with reduced penetrance and variable expressivity, predict vast differences in phenotypical presentation of situs anomalies.

**Figure 2.** Pathway of visceral left–right determination in the vertebrate.

#### **4. Left–Right Asymmetry of the Neurocognitive System**

Like the visceral organs, our mental organs, by which we mean the biological substrates of cognitive functions, are asymmetrically represented in the brain. The advantages of hemispheric functional lateralization are explained in terms of improved parallel processing and the avoidance of useless duplications that saves neural space and evades competition between redundant control centers [46,47]. In addition to a bias favoring an asymmetric brain functional organization, there is also a bias toward a prototypical asymmetric configuration at the level of the population. Most humans have their left hemisphere in charge of language, manual dexterity (giving rise to handedness), and praxis (learned gestures), and the right hemisphere in control of spatial attention, face recognition, and prosody of speech [2]. The asymmetric arrangement gives rise to functional segregation

between the left and right hemispheres. The existence of a population bias for exactly this configuration suggests that it may possess a biological advantage, but it remains to be explained why and how this would be the case. One possible way to look into this is by investigating alternative configurations of brain organization and explore their relationship with behavior.

#### *4.1. Phenotypes of Brain Functional Organization*

Recently, we have argued for the existence of three major categories in the phenotypes of functional brain segregation: typical, reversed typical, and atypical functional segregation [2]. Evidence for this distinction comes from studies investigating the asymmetry of more than one function in the same individuals. In random sample studies, this is achieved by investigating a random sample of the population [2]. The results of the available random sample studies are summarized in Table 1. Most studies probed two asymmetric functions. All used a language task as a typically left hemispheric function, and most used a spatial task to investigate right hemisphere dominance. Results reveal that most people show typical lateralization of the investigated functions and that a (substantial) minority of about 30% does not conform to this typical pattern (though many studies oversampled left-handers, which may have boosted this prevalence estimate). In about 20% of the participants, usually segregated functions were lateralized in the same hemisphere, a condition called crowding as the hemisphere is more crowded with functional representations. In about 10% of the participants, all investigated functions were lateralized in the atypical hemisphere resulting in a mirrored image of the prototypical functional segregation [2]. Evidence that this mirrored pattern of functional segregation extends beyond two atypically lateralized functions comes from selective sample research. In this type of investigation, participants are recruited based on the atypical lateralization of one function (usually language) to probe the lateralization of other functions. All these studies have been performed in left-handers as they are known to have a higher prevalence of atypical language dominance and revealed a concomitant reversal of the other investigated function [48–50]. In a recent study, five different lateralized functions were tested, and about 80% of the participants that had atypical language lateralization demonstrated complete or near complete reversal of all other functions as well [51]. In the remaining 20%, typical (or reversed typical) functional segregation was compromised more substantially, with two functions showing atypical lateralization, while the other three functions had conventional lateralization [51].

**Table 1.** Random sample studies that investigated more than one lateralized function in the same individuals.


\* Many "random-sample" studies included a proportionally higher number of left-handers to explore the effect of handedness; \*\* fTCD: functional transcranial Doppler ultrasonography; fMRI: functional magnetic resonance imaging.

#### *4.2. Reversed Typical Functional Segregation*

Together, these data confirm typical functional segregation in the majority of people, but they also show that alternative arrangements are not uncommon [2]. One alternative phenotype is a mirror reversal of typical functional segregation, which so far has been documented exclusively in left-handers [51]. Brain-wise, the reversed typical segregation phenotype is somewhat comparable with the visceral anomaly of situs inversus totalis, although its human population prevalence seems at least 100 times higher. Most random sample studies found no correlation between the laterality of different functions, suggesting that functions lateralize independently from other functions' laterality. Independent lateralization seems difficult to reconcile with a complete or near-complete reversal of five asymmetric functions in the same individual, let alone in 80% of a selective group. The odds that five independently lateralizing functions would each assume dominance in the atypical hemisphere in the same individual is extremely small. One way of reconciling independent lateralization and the observation of reversed typical functional segregation is achieved by assuming the existence of a generic blueprint of functional brain organization. Functions can develop their degree of lateralization more or less independently from other functions, but the origin of this process is seeded in a directional building plan that, on rare occasions, seems to have been flipped [2]. This assumption can explain the phenotype of the mirrored mind (mens inversus totalis, from mens, mentis (Latin) meaning mind) and at the same time allows for the independence of functional laterality indices. The assumption also predicts that the frequency by which functions occasionally deviate from the standard pattern (crowding) is not very different between the typical and reversed typical conditions as both mechanisms (independency of lateralization degree and reversal of the directional blueprint) are likely to be unrelated.

#### *4.3. Atypical Functional Segregation*

A second alternative phenotype groups conditions that show a more chaotic pattern of lateralization, as seen in individuals that have some functions showing typical and others showing atypical asymmetry. In these cases, the habitual functional segregation seems to be lost [2]. The visceral homolog of this phenotype category that we termed atypical functional segregation seems more akin to heterotaxy, where a loss of left–right asymmetry in the arrangement of the visceral organs is assumed, and that presents vast individual differences in organ displacement. While this comparison may seem farfetched at first, it has been raised before in the context of dissociated functional laterality [60], and there are more similarities between both conditions than meet the eye: variability of presentation, functional impact, and isomerism. As described above, the individual presentation of heterotaxy is very diverse, and the same gene mutation may cause severe heterotaxy affecting different organs in one individual and isolated cardiovascular malformation with no other visceral abnormalities in another. Similarly, atypical functional segregation can result from one or multiple functions deviating from the prototypical constellation [51]. Heterotaxy impacts the relationship between organs and is associated with more frequent and more severe medical problems than is situs inversus. Likewise, we reported evidence that healthy participants who show increased deviation from standard brain functional segregation perform significantly worse on a neuropsychological test battery compared to participants with typical or reversed typical segregation, suggesting that atypical functional segregation may be cognitively disadvantageous [51,61]. Finally, heterotaxy, at least theoretically, presents as two possible categories or isomerisms that copy-mirrors the left or right visceral morphology to both sides of the body. The brain functional homolog of this manifestation might be bilateral functional representation. Although bilateral functional representation has not been investigated at a multifunction level, it has received some attention at the single-function level. Research has shown that a small group of right and left-handers do not show clear-cut lateralization for language [62]. This group is said to have mixed or bilateral representation for language. Analyzing the left and right hemispheric activation patterns of these participants with a machine learning approach

distinguished participants with a bilaterally dominant language representation from those with a bilaterally non-dominant pattern [63]. These findings are in line with observations from pre-surgical Wada-testing where some patients show speech arrest following sedation of either hemisphere, and other patients do not show speech arrest following sedation of either hemisphere [64–66].

In summary, alternative organizations of hemispheric functional segregation can be distinguished in two broad phenotypical categories that show at least some common properties with the main phenotypical subgroups of visceral anomalies. It remains to be determined whether these similarities are merely the product of the finite set of options imposed by our categorization or whether they reflect more fundamental principles that share a biological mechanism.

#### **5. Left–Right Brain Development**

#### *5.1. Neurulation*

The origin of brain symmetry breaking remains to be determined, but here too, an uneven distribution of molecules is believed to initiate left–right patterning [67]. During gastrulation and opposite to the primitive streak, the ectodermic tissue thickens and flattens to become the neural plate (about 19 days postovulatory). During that stage, the notochord appears below where the primitive streak and node used to be in the mesodermic tissue, and which will induce the start of neurulation. Neurulation is the process where the ectodermal neural plate folds into a neural tube (about 25 days postovulatory). The neural tube will later develop into the central nervous system (CNS). Primary cilia are involved in neurulation by neural tube patterning and closure through regulation of Sonic hedgehog signaling, and also in neural stem cell pool regulation, neural differentiation, and migration [68]. During neural tube development, its most ventral part, adjacent to the notochord, becomes the floor plate, and its dorsal part becomes the roof plate. The floor and roof plates, respectively, project ventralizing (*nodal*, *lefty*, *shh*) and dorsalizing (bone morphogenetic protein (*bms*) that suppress default neural differentiation and instead promotes epithelial growth) inductive signals to the developing neural tube, of which its most rostral part will develop into the forebrain (Figure 3). Asymmetric secretion of morphogens from the floor and roof plates to the left and right sides of the neural tube is believed to break the symmetry of neural patterning and induce asymmetric expression of downstream genes [67,69]. In addition to the floor and roof plates, the most rostral part of the neural tube has a third patterning center, the anterior neural ridge. The anterior neural ridge is a major organizing center that emits rostralizing signals essential for developing the secondary prosencephalon (that will form telencephalon, thalamus, hypothalamus, and epithalamus) [67,69]. It has been suggested that the asymmetric expression of morphogens secreted from this region could reflect asymmetrical topographic mapping of functional regions in the cortex [70,71].

**Figure 3.** Changes during neurulation of the anterior neural section. Reprinted with permission from [72] and modified.

#### *5.2. Asymmetric Development of the Central Nervous System*

Empirical data on the asymmetry of gene expression in the left and right forebrains and midbrains of human embryos are available from 5 post-conception weeks onward [73]. By pooling data from voluntary medical abortions of healthy pregnancies and the Human Developmental Biology Resource (UK), the authors observed transcriptomic laterality in the anterior CNS regions of embryos between 5 and 14 weeks after conception. By joining the anterior CNS data with previous results of the midbrain and spinal cord regions of 4 to 8 week-old human embryos, the authors further reported evidence of age-dependent laterality of transcriptomic profiles for most structures indicating subtle differences in maturation rates between left and right CNS structures [73,74]. While both sides go through the same general developmental changes, one side appears to lead the other side at certain stages, and the laterality of the faster side is different from structure to structure. At 5 to 5.5 weeks post-conception, the spinal cord shows faster maturation on the left side than on the right, while the opposite pattern is observed for the midbrain and hindbrain [74]. By 7.5 weeks post-conception, the left choroid plexus, basal ganglia, diencephalon, and temporal cortex show faster maturation rates, but the rest of the cerebral cortex matures faster on the right side [73]. The observation of an early and differentiated pattern in the asymmetry of CNS structures with different functional destinations has led the authors to propose that brain asymmetry may be initiated/amplified at multiple locations [73]. For example, if faster maturation of the left spinal cord reflects observations of predominant right arm movements at 8 weeks post-conception (that is, prior to the innervation of the descending corticospinal tracts into the spinal cord), this could set the stage for the later cortical laterality of handedness, but would not necessarily influence the laterality of other functions or regions [73]. This suggestion is consistent with the weak correlations between the adult laterality of different brain functions like handedness and language [62] and with the results of gene ontology analysis that support the idea that handedness and language lateralization are ontogenetically independent phenotypes [75]. While subtle brain asymmetries in gene expression are already measurable at 5 weeks post-conception (i.e., approximately 7 weeks of gestational age), structural human fetal brain asymmetries become visible with current methods by the 11th week of gestational age for the choroid plexus [76], by the 16th week for the fetal cortex volume [77], by the 18th week for temporal lobe morphology [78], by the 20th week for sulcal folding [79,80], and by the 26th week for perisylvian hallmarks that have been associated with language [81]. The gap between genetic and morphological or functional brain asymmetries remains to be detailed [60].

#### **6. Are Asymmetries of Visceral and Brain Development Related?**

Visceral and neural patterning commence in close temporal proximity during the third and fourth week of human gestation, but it remains unclear whether the mechanisms that regulate visceral asymmetry also impact brain asymmetry. Asymmetric gene expression and the role of cilia seem potentially important factors for a link between visceral and brain manifestations of asymmetry.

Although *nodal* and *shh* pathways are also expressed during neurulation (cfr. floor plate induction), none of the reported 27 genes found to be differentially expressed in the left and right hemispheres of 12–14-week-old human fetal brains have known essential roles in visceral organ asymmetry [70]. Similar findings of lateralized gene expression with the more modern technique of transcriptomic profiling in post mortem temporal cortex from embryo to old age were reported, but here too, none of the reported genes have been associated with visceral anomalies [82]. On the other hand, relative hand skill in a cohort of individuals with a reading disability was associated with a variant in the gene *pcsk6*, an enzyme that cleaves *nodal* into an active form [83]. *Pcsk6* knockout mice display heterotaxy, and human variants of this gene are associated with heterotaxy and situs inversus as well, suggesting that handedness is at least in part controlled by genes that contribute to the determination of visceral asymmetry [83]. Human genes, like *GPC3*, associated (though not

significant at a genome-wide threshold) with relative hand skill in the general population, cause situs anomalies when their orthologs are knocked out in mice [83,84].

Clinical evidence demonstrates the importance of cilia in human neurulation. Major ciliopathy-associated hereditary cerebral anomalies include neural tube defects, corpus callosum malformations, cerebellar hypoplasia, and hydrocephaly. Less severe neurological features, including cognitive deficits, autism spectrum disorders, and seizures, are also frequently observed in individuals with ciliopathies and hint at the possibility of more subtle cortical deficiencies [68]. Concerning laterality, genes most strongly associated with relative hand skill in a dyslexia cohort are involved in ciliogenesis, and their disruption in mice causes situs inversus [83,84]. In addition, cilia-related gene sets are more highly expressed in the right choroid plexus in the 7.5–13 post-conception age range [73]. The choroid plexus is also the first brain structure showing morphological asymmetry and is associated with the circulation of cerebrospinal fluid in the ventricles. Despite these observations, there is no clear evidence that cilia play a role in the initiation or propagation of central nervous system asymmetry [73].

#### **7. Atypical Brain Asymmetry in Human Visceral Situs Anomalies**

As the molecular regulation of brain asymmetry and its relationship with visceral lateralization remains to be elucidated, an alternative strategy of investigation is to look for evidence of atypical functional or structural brain asymmetry in people with situs anomalies. If the prevalence of behavioral, brain functional, or brain structural asymmetry differs between participants with typical and atypical visceral situs, then research would be better informed to explore more specific pathways of a possible link between human visceral and brain asymmetry. This approach is confronted with two major limitations: sample size and heterogeneous causality. As situs anomalies are inherently rare, it is extremely difficult to recruit many participants with atypical organ situs, especially if more intensive research protocols like neuroimaging are applied. In the absence of striking relations, small samples limit the statistical power to detect more subtle differences between typical and atypical groups in particular when only a subsample of participants shows a relation and others do not. This brings us to the second limitation of this approach, the heterogeneity of factors (genetic and other) that contribute to brain and visceral asymmetries. Different manifestations of situs anomalies have been associated with different genetic mutations, suggesting that genetic screening or at least a thorough description of the situs condition and family history should be used for categorization. Many gene mutations and combinations thereof have been associated with anomalies in visceral left–right patterning, and they are known to affect different steps and mechanisms of this complex process. It is plausible that some gene mutations bear no relation with brain asymmetrization, while others do. For example, in primary ciliary dyskinesia, the resulting randomization of organ situs is due to genetic mutations causing ciliary dysfunction. While cilia have a role in neurulation, it is unclear whether this includes lateralization of morphogens that induce brain asymmetry. Hence, a ciliopathy like primary cilia dyskinesia may not affect developing brain asymmetry at all. It is also possible that in people with situs inversus that have no primary ciliary dyskinesia, the origin of their situs anomaly is due to a temporary (or local) malfunction of nodal cilia or is caused by a different mechanism altogether. Even within the subgroup of situs inversus, etiological heterogeneity is substantial and extends beyond the role of genes. This was illustrated in a recent genome sequencing study of 15 cases with situs inversus totalis (SIT) [25]. The subgroup of six participants with primary ciliary dyskinesia (PCD) all presented with likely recessive PCD-associated mutations. Similar mutations were also detected in two of the non-PCD SIT participants, and in two other non-PCD SIT participants, recessive mutations in genes linked to situs inversus outside the context of PCD were found. In five of the nine non-PCD cases, however, no monogenic basis for their situs anomaly was found, which led the authors to consider early environmental or stochastic effects as possible causative factors.

#### **8. Systematic Review**

In March 2021, we performed a systematic literature search to address whether visceral situs anomalies have a different prevalence of brain and behavioral asymmetry [85]. The following platforms were searched: Web of Science (indexes: SCI-EXPANDED, SSCI, A&HCI, CPCI-S, CPCI-SSH, ESCI; range: 1972–2021), PubMed, and Google Scholar. In all cases, we searched for articles with the following strategy 1. Topic: situs inversus OR heterotaxy; 2. Topic: brain asymmetry OR brain functional asymmetry OR brain structural asymmetry OR behavio(u)ral asymmetry OR hemispheric dominance OR brain laterality; 3. #1 AND #2. We obtained a total of 79 records (WoS *n* = 63; PubMed *n* = 11; Google Scholar *n* = 5). Sixty-nine records were screened after the removal of duplicates. Records on animal research (*n* = 25), genetics (*n* = 17), and medical papers on comorbidities or laterality defects other than situs inversus or heterotaxy (*n* = 17) were excluded. Ten full-text articles were addressed for eligibility. In the references of these articles 7 further (mostly older) studies were identified that reported empirical data on the research question. While this manuscript was under revision, an additional paper on brain asymmetry in fetuses with laterality defects was accepted for publication and added to the review [86]. A total of 18 studies were included in the qualitative synthesis for this systematic review.

Many studies have not described the situs condition of their participants in detail, nor have they differentiated their already small samples of participants into separate categories or explored their genetic background. In the next section, we will summarize the behavioral, brain structural, and brain functional data on atypical asymmetry in participants with situs anomalies or, to be more precise, in participants with situs inversus as almost all brain and behavior-related research in this field has been performed within this subgroup.

#### *8.1. Handedness in Situs Inversus*

Already in 1836, Sir Thomas Watson remarked that individuals with situs inversus (SI) are no more left-handed than the rest of the population (reported in [87]). This observation was empirically confirmed in several remarkably large-scaled studies of the early to mid-20th century (Table 2) [88,89]. These early studies, however, suffer from poor behavioral assessment of handedness and poor etiological description of the situs anomaly. In addition, the reported prevalence of left-handedness around 6–7% is clearly lower than contemporary estimates of 10% [90], suggesting that cultural pressure against left-hand use and forced right-handedness may have underestimated natural left-hand preference in these cohorts. As a result, their findings might not provide a clear answer to the question at hand. Then follow two smaller studies based on hospital samples and reporting the low prevalence of non-right-handedness in 6 and 16 SI participants, respectively [91,92]. Unfortunately, very little information on recruitment and SI status or etiology was provided. In both studies the authors concluded that there was little evidence for a relationship between handedness and visceral position. Two later studies that focused on handedness and which recruited quite sizeable cohorts included PCD-related SI participants only. Both studies came to the conclusion that the prevalence of left-handedness in PCD-related SIT is no different from the rest of the population [32,93]. Given the reports of a possible genetic association between relative hand skill and ciliogenesis, typical handedness in PCD-related SIT may seem surprising and has been explained in terms of compensatory mechanism that allow the typical development of handedness to overrule the influence of ciliopathy [83,84]. For non-syndromal SI, the issue of handedness is less clear given the paucity or incompleteness of available data. Some information can be gathered from studies that investigated brain functional asymmetry in SI and which predominantly featured non-syndromal cases of SIT (Table 3). Together, these studies report on 22 sporadic cases that were explicitly reported to be free of PCD-symptoms [94–98]. Seventeen of these participants were right-handed, and 5 were left-handed (29% left-handedness). It needs to be remarked that all left-handers were reported by the same study in which 5 out of 9 non-PCD-related SIT had a left-hand preference (55% left-handedness) [98]. Interestingly, this study also recruited 6 PCD-related SIT cases, only one of which was a left-hander, a result that was in line with previous

findings on hand preference in PCD-related SIT. Is the seemingly random hand preference in the non-PCD-related SIT participants of the Ghent-cohort an accidental finding? It may well be as none of the other studies even remotely suggested anything of the kind. Future research, preferably in a larger cohort of PCD and non-PCD-related SIT, is necessary to determine if the differential effect of situs inversus on handedness can be replicated. At the same time, the possibility that the etiology of the SI anomaly may differentially influence brain-related asymmetry underlines the importance of providing a detailed description of the SI participants' phenotype and, if possible, also take the genotype into account.


#### **Table 2.** Overview of handedness studies in situs inversus.


**Table 3.**Overview of brain functional asymmetry studies in situs inversus.

\* EHI: Edinburgh handedness inventory; \*\* DLT: dichotic listening test; fMRI: functional magnetic resonance imaging; MEG: magneto-encephalography; \*\*\* REA: right ear advantage.

#### *8.2. Brain Functional Asymmetry in Situs Inversus*

As mentioned in the previous paragraph, brain functional asymmetry was predominantly investigated in participants with a non-syndromal manifestation of SI (Table 3). The discussion starts in the late 1980s–early 1990s with the report of two right-handed stroke patients with visceral anomalies, one of which became aphasic following a left hemisphere cerebrovascular lesion [94] while the other, a patient with left isomerism heterotaxy, showed crossed-aphasia after a right hemisphere stroke [99]. More convincing evidence for typical language lateralization came from 9 SIT participants (only one with PCD-related SIT), who performed a dichotic listening paradigm and showed typical right ear advantage in all, but one case [95]. The advent of MRI research provided the opportunity of visualizing neural activation during cognitive tasks. A first fMRI study corroborated Tanaka's dichotic listening findings by showing typical left hemisphere lateralization for language in three non-syndromal SIT participants [96], but a decade later, a second fMRI study reported atypical right hemisphere lateralization for language in two out of three SIT cases [100]. Until now, all studies, including a longitudinal case study that used fMRI [97], had focused on language. Recently, research broadened to other lateralized functions, including praxis, spatial attention, and face recognition, in an fMRI study of 15 SIT participants, of which 6 had PCD-related SIT, and 9 had non-PCD-related SIT [61]. While 80% of this cohort had left hemisphere language dominance, suggesting generally typical language lateralization, a control group matched for handedness showed 93% leftward lateralization. The same trend was found for the three other tested functions that all showed more typical asymmetry in the matched controls compared to the SIT participants. The authors concluded that atypical functional segregation, that is, the likelihood that brain functional organization does not show the typical population pattern, is more frequent in SIT participants. No obvious difference in the level of deviation from typical functional segregation was observed between PCD and non-PCD-related SIT, but the small sample size limits proper statistical comparison. It can be argued that results on functional lateralization have been influenced by the unexpectedly high number of left-handers in this sample as left-handers have a higher prevalence of atypical functional lateralization [2], but atypical lateralization occurred equally frequently in the right-handed SIT participants. Together, the available data suggest that, while most people with SIT will show typical patterns of functional asymmetry, atypical lateralization of language and other asymmetric functions may be more frequent in SIT. It remains to be determined whether this is a general trend or associated with specific etiological characteristics.

#### *8.3. Brain Structural Asymmetry in Situs Inversus*

An overview of studies reporting on brain structural asymmetry in SI is provided in Table 4. If there is one consistent finding on brain asymmetry in SI, it is the observation that their cerebral torque is generally reversed than the typical human population bias. The cerebral or "Yakovlevian" torque is a gross anatomical and morphologically complex characteristic [101] that refers to an anti-clockwise twist of the brain about the ventraldorsal axis. It is most often described in terms of its petalia, whereby the right frontal pole protrudes anteriorly to the right frontal pole, and the left occipital pole protrudes posteriorly to the right occipital pole. Typical petalia asymmetry is observed in 44% of modern human brains [102] and appears to be absent in non-human primates [101]. Reversed petalia were reported in 15 out of 23 SIT participants (65%), most of which were sporadic cases. Again, a possible distinction arises between syndromal and non-syndromal SIT as a recent study documented complete reversal of the petalia in 7 out of 9 non-PCD-related SIT participants (78%) and in none of the 6 PCD-related SIT participants [98]. It remains to be confirmed whether the reversed cerebral torque pairs with the reversal of intracranial vasculature and bony landmarks as suggested by one post-mortem study [103]. If it does, it would be an important argument for a link between different brain morphological asymmetries and a link between lateralized gradients of brain structural and visceral development.


**Table 4.**Overview of studies on brain structural asymmetry in situs inversus.

\* MRI: magnetic resonance imaging; \*\* IFG: inferior frontal gyrus; STS: superior temporal sulcus.

No systematic reversals in other structural brain asymmetries have been reported in SI. Alleged language-related markers like the planum temporale, Sylvian fissure, inferior frontal gyrus, depth of the superior temporal sulcus, and the arcuate fasciculus show the same variability and directional bias as the general population. The available data are scarce, though, and the discovery of more subtle effects or between SIT-type differences awaits further research.

#### **9. Discussion**

The low prevalence and substantial phenotypical variability of human visceral laterality anomalies postpone consensus on clear classification criteria for subgroup determination. Still, two broad categories of anomalies are generally distinguished, *situs inversus* characterized by a complete or near-complete mirror reversal of typical visceral asymmetry, and *heterotaxy* described as a duplication of one of either asymmetric sides. Both phenotype categories are believed to result from different deficits in the complex developmental cascade of visceral left–right differentiation, but the exact causal implications for each step and each genetic mutation in that process remain to be elucidated. The same is true for the brain. While the prevalence of nonconventional brain organization is roughly 100 times more frequent than atypical visceral organization, it is more difficult to assess, and data are scarce. However, here too, two main categories of unconventional brain organization are advanced, *reversed functional segregation* presenting as a mirror image of the usual hemispheric task division, and *atypical functional segregation* characterized by functional crowding.

The substantial difference in the prevalence of atypical visceral and brain organization also brings the effect of evolutionary canalization to mind, the increased resistance of a trait to genetic and environmental perturbations over evolutionary time. Left-sided heart anatomy is a preserved trait in all living vertebrates, but the incidence of spontaneous reversal declines throughout vertebrate evolution from 5% in fish, 1–2% in amphibians, 0.1% in mammals, and 0.01% in humans [9]. Explanations for the evolutionary increase in canalization include increased predictability of symmetry breaking by cilia-controlled nodal flow or the more stable conditions of the placental environment [9]. Cladistic estimates of reversals in brain organization are not available, but the concept of evolutionary canalization may provide an interesting venue to explore the origin and timing of brain structural and functional asymmetries in humans by comparing prevalence measures of atypical laterality.

Apart from similarities in the overall appearance of the main phenotype subgroups of visceral and brain laterality anomalies, we should keep in mind that the visceral and neural systems serve fundamentally different biological functions and that the reasons for developing asymmetry in each system are likely to be dissimilar. Nevertheless, selfsame basic mechanisms for left–right differentiation may be employed by both systems to generate and/or propel asymmetry [9]. This possibility is hinted at by some mutant lines in vertebrate species that appear to align atypical visceral with atypical brain structural asymmetry and which also appears to impact their behavioral asymmetries [3,5]. One way to explore such a relation in humans is to investigate and compare the developmental cascades of visceral and brain laterality and scrutinize the molecular genetics underlying both mechanisms for biological links or similarities. The road toward asymmetry appears very complex and much of it, particularly concerning the brain, remains to be discovered.

An alternative way to explore possible relations lies in the direct comparison of phenotypes by investigating brain and behavioral asymmetries in individuals with situs anomalies. Delineation of atypical manifestations could provide molecular genetics with more specific targets to find associations between the developmental cascades of visceral and brain asymmetry. While this approach is hampered by the low prevalence of situs anomalies and the laborious assessment of brain asymmetries, several studies have contributed to this endeavor. However, samples are often small, and the range of phenotypes is restricted or poorly defined.

Most, if not all, studies on behavioral and brain asymmetry in situs anomalies focused on situs inversus (totalis). Probable reasons for this selective approach are the anticipation of more straightforward results and the better medical condition of participants with situs inversus compared to those with heterotaxy. In general, the studies appear to agree that situs inversus in humans is not inseparably associated with a reversal of brain and behavioral asymmetries as seen in some other species. On the contrary, most people with situs inversus seem to present with typical patterns of hemispheric specialization, although a higher prevalence of functional crowding in this group remains a possibility. At the same time, the findings hint at some more subtle effects that distinguish between types of situs inversus with different etiologies. More in particular, in PCD-related syndromal situs inversus, handedness and probably also brain torque reveal the same laterality bias as the general population. This finding can be taken to suggest that nodal ciliopathy and the eventually reversed subsequent molecular cascade that gives rise to visceral laterality has only little effect on hand preference and gross brain morphology. By contrast, situs inversus caused by non-ciliary, perhaps earlier, factors does seem to be accompanied by a reversal of the brain torque. This finding is reported by several independent studies from North America, Japan, and Europe and indeed hints at a possible relation between human visceral asymmetry and the asymmetrical shape of the brain organ. A possible venue to investigate a direct relation between both manifestations of directional asymmetry in humans would be to determine signed fluctuating asymmetry of the visceral and brain torque modules in a sample of humans, which do not necessarily need to have a visceral anomaly [8]. To corroborate and extend findings on brain asymmetry in visceral anomalies, future research should provide detailed phenotypical information of participants supplemented by genetic data if possible. Ideally, a consensus should be reached on core information to be reported that will allow open science and meta-analytic initiatives to gather larger samples of participants with situs anomalies and further understand possible interactions between human visceral and brain asymmetry.

**Author Contributions:** G.V. wrote the manuscript with support from R.G. and H.V. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable (review).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Glossary**

**Anterior neural ridge**: The anterior neural ridge is a region in the neural plate and later neural tube, which secretes signaling molecules essential for developing the forebrain. **Antisymmetry**: Dextral and sinistral forms are equally present within a population. **Atypical hemispheric functional segregation**: Phenotype of hemispheric functional segregation in which the typical left–right segregation is lost due to one or more functions showing atypical dominance, while other functions do not. **Autosomal dominant disorder**: A pattern of inheritance in which an affected individual has one copy of a mutant gene and one normal gene on a pair of autosomal (one of the numbered, non-sex) chromosomes. **Autosomal recessive disorder**: A pattern of inheritance in which an affected individual requires two copies of a mutant gene on a pair of autosomal (one of the numbered, non-sex) chromosomes. **Behavioral asymmetry**: Left–right difference in behavior, like hand or foot preference, or the increased probability to retain words presented to the right ear versus those presented to the left ear. **Brain asymmetry**: Left–right differences in functional or structural (anatomical) characteristics between the two hemispheres. **Canalization (evolutionary canalization)**: Increased resistance of an established trait for genetic or environmental perturbations over evolutionary time. **Cascade capture**: The recruitment of genes or gene cascades for another duty. **Cilium/Cilia**: Small hair-like organelles that protrude from the larger cell body. Cilia can be motile or non-motile. Non-motile cilia serve as sensory organelles, much like a cellular antenna. Cells of the transient primitive node have singular motile cilia known as nodal cilia, critical for the establishment of left to right body asymmetry. **Ciliogenesis**: The building of the cell's cilium/cilia. Defects in ciliogenesis can lead to numerous human diseases related to non-functioning cilia (ciliopathies). **Ciliary motility**: The ability of some cilia types to produce motion by a molecular motor that drives its beating. Motile cilia have a function in the transport of fluids over the surface of cells. **Dextrocardia**: A rare congenital condition in which the heart's apex is located on the right side of the body. **Directional asymmetry**: Most individuals in a population are asymmetrical in the same direction (population bias). **Floor plate**: Located on the ventral midline of the embryonic neural tube, the floor plate is a glial structure that serves as an organizer to ventralize tissues in the embryo as well as to guide neuronal positioning and differentiation along the dorsoventral axis of the neural tube. **Fluctuating asymmetry**: The amount of deviation from perfect bilateral symmetry as reflected by small differences between the left and the right sides due to random errors in the individual development. **fMRI**: Functional magnetic resonance imaging is a non-invasive technique to measure and map changes in the brain's blood flow that coincide with brain activity. **Forebrain (prosencephalon)**: The rostral (forward-most) portion of the brain that will develop into the diencephalon (thalamus, hypothalamus, subthalamus, and epithalamus) and the telencephalon, which develops into the cerebrum. **fTCD**: Functional transcranial Doppler ultrasonography is a non-invasive technique to measure changes in the blood flow velocity of the basal segments of the cerebral arteries that coincide with brain activity. **Gastrulation**: A phase in early embryonic development during which the single-layered hollow sphere of cells (blastula) is reorganized into a multilayered structure (gastrula). By the end of gastrulation, the embryo has begun differentiation to establish distinct cell lineages and set up the basic axes of the body. **Genetic assimilation**: An alternative mechanism of variation (compared to mutations, in which genotype precedes phenotype) in which developmental plasticity creates novel phenotypes before heritable variation exists (phenotype precedes genotype). Genetic control over the new phenotype arises later through random mutations. **Genotype**: The particular type and arrangement of genes of an organism. **Hemispheric dominance**: The phenomenon that cognitive processes tend to be specialized to one side of the brain or the other, as demonstrated by aphasia following left hemisphere lesions and spatial neglect following right hemisphere lesions in most people. **Hemispheric functional segregation**: The division of labor in cognitive tasks between both hemispheres. In humans, hemispheric functional segregation shows a strong population bias toward prototypical segregation in which the left hemisphere is known to be dominant for language, fine motor control, and praxis (learned gestures), whereas the right hemisphere supports spatial attention, face recognition and prosody of speech. **Heterotaxy**: The loss of typical left–right laterality in the arrangement of the visceral organs along the superior–inferior axis, also referred to as situs ambiguus. **Kartagener syndrome**: A rare, autosomal recessive genetic ciliary disorder comprising the triad of situs inversus, chronic sinusitis, and bronchiectasis. **Lateral plate mesoderm**: A type of mesoderm that is found at the periphery of the embryo. *Lefty*: A class of proteins related to the superfamily of growth factors that play a role in left–right asymmetry determination of organ systems during development. **Levocardia**: A condition where the heart is on the left (typical) side of the thoracic cavity. **Neural tube**: The embryonic precursor to the central nervous system, which is made up of the brain and spinal cord. **Neurulation**: The folding process in vertebrate embryos, which includes the transformation of the neural plate into the neural tube. *Nodal*: A protein that is encoded by the human *NODAL* gene, which belongs to the transforming growth factor-beta superfamily. It is involved in cell differentiation in early embryogenesis, playing a key role in signal transfer from the primitive node, in the anterior primitive streak, to the lateral plate mesoderm. **Nodal flow**: The (leftward) movement of fluid at the prim-

itive node caused by ciliary movement and taken to be a central process in symmetry breaking on the left–right axis. **Ortholog**: A homologous gene found in different species related by linear descent. **Phenotype**: The sum of an organism's observable characteristics or traits. *Pitx2*: A protein that in humans is encoded by the *PITX2* gene. This protein acts as a transcription factor and is involved in developing the eye, tooth and abdominal organs. **Pleiotropy**: Occurs when one gene influences two or more seemingly unrelated phenotypic traits. Mutation in a pleiotropic gene may affect several traits simultaneously. **Primitive node**: The organizer for gastrulation in the vertebrate embryo. **Primary ciliary dyskinesia**: A rare, ciliopathic, genetically heterogeneous disorder that causes defects in the action of cilia lining the respiratory tract (lower and upper, sinuses, Eustachian tube, middle ear), fallopian tube, and flagellum of sperm cells. **Reversed typical hemispheric functional segregation**: Phenotype of hemispheric functional segregation in which the left–right laterality of functions is reversed than the typical organization seen in the population. While the habitual functional segregation is maintained, the phenotype is a mirror image of the usual functional brain organization. **Roof plate**: An embryonic organizing center consisting of specialized glial cells that occupy the dorsal midline of the vertebrate neural tube. The roof plate generates morphogenic signals along the length of the neuraxis, which control the specification and differentiation of dorsal neuronal cell types. *Shh*: Sonic hedgehog (Shh) is a protein that, in humans, is encoded by the *SHH* gene. *Shh* plays a key role in developing many animals. In vertebrates, it is involved in organogenesis. **Signaltransducing pathway**: Signal transduction is the process by which a chemical or physical signal is transmitted through a cell as a series of molecular events, which ultimately results in a cellular response. The changes give rise to a chain of biochemical events known as a signaling pathway. **Situs ambiguus**: Medical term referring to a loss of the typical left–right positioning of thoracic and abdominal organs, also called heterotaxy. **Situs inversus (totalis)**: Medical term referring to a reversal of the typical position of thoracic and abdominal organs. **Situs solitus**: Medical term referring to the typical position of thoracic and abdominal organs. **Transcriptomics**: The study of the transcriptome—the complete set of RNA transcripts that are produced by the genome—using high-throughput methods, such as microarray analysis. **Typical hemispheric functional segregation**: Phenotype of hemispheric functional segregation that, due to a population bias, is most common in the human population. **Visceral asymmetry**: Refers to the asymmetry in left–right positioning of thoracic and abdominal organs.

#### **References**


## *Review* **How Asymmetries Evolved: Hearts, Brains, and Molecules**

**Michael C. Corballis**

School of Psychology, University of Auckland, Auckland 1142, New Zealand; m.corballis@auckland.ac.nz; Tel.: +642-211365674

**Abstract:** Humans belong to the vast clade of species known as the bilateria, with a bilaterally symmetrical body plan. Over the course of evolution, exceptions to symmetry have arisen. Among chordates, the internal organs have been arranged asymmetrically in order to create more efficient functioning and packaging. The brain has also assumed asymmetries, although these generally trade off against the pressure toward symmetry, itself a reflection of the symmetry of limbs and sense organs. In humans, at least, brain asymmetries occur in independent networks, including those involved in language and manual manipulation biased to the left hemisphere, and emotion and face perception biased to the right. Similar asymmetries occur in other species, notably the great apes. A number of asymmetries are correlated with conditions such as dyslexia, autism, and schizophrenia, and have largely independent genetic associations. The origin of asymmetry itself, though, appears to be unitary, and in the case of the internal organs, at least, may depend ultimately on asymmetry at the molecular level.

**Keywords:** bilateria; cerebral asymmetry; handedness; language; molecular asymmetry; situs

**Citation:** Corballis, M.C. How Asymmetries Evolved: Hearts, Brains, and Molecules. *Symmetry* **2021**, *13*, 914. https://doi.org/10.3390/ sym13060914

Academic Editors: Sebastian Ocklenburg, Onur Güntürkün and Chiara Spironelli

Received: 15 April 2021 Accepted: 17 May 2021 Published: 21 May 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the author. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

#### **1. The Symmetrical Background**

The evolution of asymmetry should be understood in relation to its opposite, the overwhelming bilateral symmetry which characterises the vast clade of organisms to which we belong. These are the bilateria. They go back at least to the Cambrian, beginning some 541 million years ago, and probably slightly earlier into the late Protozeroic [1]. Bilateral symmetry emerged in species that move in space, and depends on the prior establishment of two bodily axes. The antero–posterior axis may have arisen first in relation to feeding, involving openings at head and tail separated by a through-gut [2], as in worms that burrow. The demands of locomotion led further to sense organs, such as eyes and nose, oriented toward the direction of motion, and the limbs were shaped to facilitate linear motion in a consistent direction, further defining the antero–posterior axis. The dorsal–ventral axis evolved later through the influence of gravity and the demands of locomotion, creating consistent differences between top and bottom, such as eyes placed high for distance vision and feet touching the ground. The formation of these two axes, with their distinctive asymmetries, appears to be highly conserved genetically, at least across vertebrates and arthropods [3].

Only when these two axes are established can the left–right axis be defined, and the body remains highly symmetrical along this axis. The great British scientist Sir Isaac Newton remarked that this symmetry, with the exception only of the bowels, proved "the counsel and contrivance of an Author." There is no need, though, to appeal to a deity; bilateral symmetry can be understood in evolutionary terms. As an animal moves around, the environment it encounters is largely indifferent to whether things are on the left or right. Predators and prey and obstructions to movement can occur on either side. With respect to movement and orientation in space, there seem to be no contingencies favouring differences between the left and right sides of animals.

Bilateral symmetry, though, is not merely a matter of default; it also enhances biological fitness. In animals that move freely, locomotion is almost universally dependent

on paired limbs, be they legs, flippers, or wings, and symmetry ensures linear movement, which provides the most efficient way to journey between two points. Having one leg longer than the other, or functionally more efficient, might leave an animal moving in circles, or at least making multiple corrections. Animals also need to be as sensitive to features on their left as on the right if they are to respond optimally to danger or to exploit what the environment has to offer. This means that sense organs, such as eyes, ears, and skin receptors, are symmetrically placed.

Much of behaviour is a matter of programming movement or processing information provided by the senses, creating evolutionary pressure for the brain itself to be symmetrical. Indeed, for much of human history the two sides of the brain were considered duplicates, albeit mirror images. Descartes [4], for example, observed "the brain to be double" (p. 275). In terms of gross anatomy, at least, the left and right sides did seem to be mirror images, causing the French physician Marie Francois Xavier Bichat (1771–1802) to formulate the "law of symmetry." Bichat died at the age of 30 and was not widely known at the time, but his law of symmetry gained wide currency in the 19th century, especially through the influence of Franz Joseph Gall (1758–1828) [5].

There is even pressure for the brain to preserve its bilateral symmetry in the face of asymmetrical experience. It is well established that animals have more difficulty learning to distinguish left–right mirror images than up-down mirror images, and tend to treat left–right mirror images as though they were the same [6]. In one classic experiment, people shown 2500 pictures were later able to recognise them with surprising accuracy, except that they were as likely to report a picture as familiar when it was the left–right reverse of the original as when it was the original itself [7]. So-called left–right equivalence is especially evident in young children learning to read; up until the age of six or so, they frequently write letters or words backwards, despite being shown them only in correct orientation [8]. Animals, too, have much more difficulty discriminating left–right mirror images than in discriminating up-down mirror images. Left–right equivalence is adaptive in the natural world, where objects or animals can occur in opposite profiles, and events on one side of the body might next time occur on the other side.

The equivalence of left–right mirror images can be attributed to a process of interhemispheric reversal in the formation of memories, so that memories are held both in the veridical format and the mirrored one [9]. Logothetis et al. [10] found that some single cells in the inferotemporal cortex of two adult rhesus monkeys responded equivalently to meaningless mirror-image shapes, and remarked that "Distinguishing mirror images has no apparent usefulness to any animal" (p. 360). It can be an impediment, though, in some human activities, notably in learning to read and write scripts written in a consistent left–right direction. With specific training, it can be overcome. Torres et al. [11] found that three weeks of training first-grade children to discriminate mirror-image letters, such as b and d, led to a doubling of reading speed: "a simple and cost-effective way to unleash the reading fluency potential of millions of children worldwide" (p. 742).

#### **2. Emerging Asymmetries**

#### *2.1. Internal Organs*

Bilateral symmetry, then, is a striking feature of nearly all animals, but there are also longstanding asymmetries. The most extreme example is situs solitus, the asymmetrical placement of thoracic and abdominal organs. In the vast majority of humans, for example, the heart is displaced to the left, along with the stomach, spleen, and aorta, while the liver, gall bladder and trilobed lung are displaced to the right. Approximately one in 10,000 individuals have situs inversus totalis, in which these asymmetries are reversed [12] and where it does occur, it seems to arise as a matter of chance when the normal directional influence is lacking [13,14]. The asymmetries are fundamentally the same in all vertebrates [15], and more generally in chordates, suggesting that they have a common origin and go far back in evolution [16].

In vertebrates, at least, imposing asymmetry on internal organs is adaptive. For example, a mass of muscle such as the heart, achieves much greater efficiency of pumping from a spirally coiled form than from a simple tube [17]. Beyond that, it is probably essentially a matter of efficient packaging in the human body. Just as it would be inefficient to pack a suitcase while maintaining perfect symmetry of the contents, so it is that the internal organs are arranged asymmetrically in the body. Similarly, design of an automobile abandons symmetry in its internal engine and controls, while largely maintaining symmetry of external body shape. Manufacturing has adopted a design long evident in biological evolution.

Deviations from bilateral symmetry can occur through random influences—no animal is perfectly symmetrical, even discounting the asymmetries of the internal organs. However, reliance on random or fluctuating asymmetry for internal organs would run the risk of error, so consistent asymmetry was stamped in early in evolution. Situs solitus, is clearly an ancestral condition, and is all but universal. Bilateral symmetry and asymmetry therefore coexist in a trade off, with pressure toward one vying with pressure toward the other.

#### *2.2. Handedness*

The clearest evidence of a trade off comes from use of the hands or forelimbs, which in some species is symmetrical while in others there seems a clear species-wide preference for one or the other in certain actions. For most animals, bilateral symmetry of the limbs is adaptive, especially in movement, but in bipedal animals the forelimbs are freed from locomotion and are potentially open to specialization. Symmetry of action can still be adaptive in reaching and grasping with the hands, but in more complex actions, biological fitness may benefit if the hands adopt complementary roles, such as one hand holding an object while the other operates on it. In some cases, one hand assumes a dominant role. For example, bipedal marsupials, such as kangaroos, show a 90 percent preference for the left hand when feeding, whereas quadrupedal marsupials, such as the sugar glider or grey short-tailed opossum, show no preference at the population level [18]. Cats and dogs, too, show no bias at the population level, but individual animals often show a consistent preference for one or other paw in activities such as reaching [19]. (For a more general review of limb preferences in non-human vertebrates, see [20].) Our closest nonhuman relatives, chimpanzees, are less consistently bipedal than are we humans, and correspondingly show lower right-hand preference, at approximately 65–70 percent, in intricate manual actions [21]. Gorillas are predominantly right-handed in bimanual actions, where the non-dominant hand holds a food-related object and the dominant hand performs actions on it, such as dipping, stripping, or extracting [22].

At least one study has shown a slight right-hand advantage for rhesus monkeys but no bias in capuchins [23]. It is not restricted to primates; for example, some 77% of walruses display a preference for the right flipper when feeding [24]. Some creatures, though, are clearly left-handed—or left-"limbed." In some species of parrot, approximately 90% of individuals show a preference for using the left foot when picking up pieces of food [25], and as we have seen bipedal kangaroos are predominantly left-handed. The preference for one or other limb being dominant is seldom if ever absolute, with the dominance ranging from approximately 65 to approximately 90 percent [26].

In humans, bipedalism is obligate and the hands are correspondingly less involved in locomotion and more available for asymmetrical activities such as tool manufacture, throwing, and writing, in all of which the right hand is dominant in some 90 percent of the population. Yet, the symmetry between the hands is largely preserved in their basic anatomy as well as in simple operations, such as reaching and grasping, and even catching. People can intercept a moving object equally well with either hand, but throw much more efficiently with just one hand, usually the right [27]. Most cricketers or baseball players, for example, can make one-handed catches with either hand, but few can throw even adequately with the non-preferred hand. The trade off between symmetry and asymmetry is therefore well illustrated in the way we use our hands.

#### *2.3. Cerebral Asymmetry in Humans*

Perhaps the first intimation of an exception to the law of symmetry as applied to the brain arose at a meeting in Montpellier in 1836, when an obscure French physician called Marc Dax produced evidence that speech was localised in the left hemisphere. This was largely disregarded, but some twenty-five years later, a more eminent physician called Paul Broca [28,29] showed that speech was disrupted following damage to the portion of the left prefrontal cortex since labelled as Broca's area, confirming the lefthemispheric dominance for speech. At that point, Dax's son recognised the significance of his father's work and arranged to have the early manuscript published, along with further evidence from 140 patients [30]. Evidence also emerged that comprehension of speech was impaired after damage in the left superior temporal gyrus, in the area since known as Wernicke's area. [31]. By the late 19th century, then, the brain was understood to exhibit some fundamental asymmetries, at least in function, in spite of its seeming anatomical symmetry. At this point, it was recognised that handedness itself was due to brain asymmetry, adding to the notion that the left hemisphere was the dominant or major hemisphere, with the right relegated to minor status. With some hesitation from the French medical establishment [32], the law of symmetry was overturned.

These developments also led to the view that the two sides of the brain were not simply uneven, but functioned in some ways as complementary opposites. In the most extreme versions, the left hemisphere was said to harness humanity, volition, masculinity, and reason, while animality, instinct, femininity, and madness were closeted in the right. This phase of speculation is well described by the historian Anne Harrington [33], who observed that it probably owed more to the social prejudices of the time than to the neurological facts. She wrote, "It is interesting that, once one has given the two hemispheres sexual identities, the idea of cerebral dominance becomes a rather apt metaphor for the social and economic domination of men over women in 19th-century Europe" (p. 624).

These extreme notions seemed to subside after the turn of the 20th century, but a second wave of speculation followed the split-brain research of the 1960s, when a series of patients underwent section of the forebrain commissures for the relief of intractable epilepsy. Again, the left hemisphere was shown to be dominant for language [34], and in 1981, Roger W. Sperry belatedly received the Nobel Prize in Physiology or Medicine "for his discoveries concerning the functional specialization of the cerebral hemispheres." There again followed a barrage of speculation about the duality of mind, with the left brain described as logical, rational and mechanistic, and the right brain intuitive, emotional and creative [35]. The social and political pressures of the time were different from those of the previous century, and the protests against the war in Vietnam, feminism, and anti-establishment movements seemed generally to anoint the right brain as favoured over the militaristic left. In his Nobel address, Sperry [36] himself noted, "The left-right dichotomy in cognitive mode is an idea with which it is very easy to run wild" (p. 1226). The dichotomy is still with us in popular culture—and indeed often runs wild.

Brain asymmetry, then, was a comparatively recent discovery in human history, and a revelation against the general assumption of bilateral symmetry. It was linked, moreover, to specifically human aspects of thought. This has led to a tendency to regard it as uniquely human (e.g., [37]), and perhaps even a species-defining feature [38]. This is also implicit in the view that language itself is unique to our species (e.g., [39]). The idea that brain asymmetry emerged only in *Homo sapiens* has no doubt dampened efforts to understand its evolutionary origins, although this has begun to change with the realisation that asymmetries are ubiquitous in biology.

It is also commonly assumed that brain asymmetry is unidimensional, to the extent that individuals are often described as being either left- or right-brained, implying that the dominance of one or other hemisphere operates as a whole. It has become clear, though, that there are several, perhaps many, dimensions of laterality. Handedness, too, is effectively a cerebral asymmetry, not a manual one, and is commonly associated with the left-hemispheric dominance for speech. The correlation is in fact much weaker than

previously assumed [40]. Some 95 percent of right-handers are left-cerebrally dominant for language, but so are 70–80 percent of left-handers [41]. Situs inversus totalis does not seem to reverse normal handedness or functional brain asymmetry, with the exception of the Yakoklevian torque—an anatomical asymmetry normally characterised as a protrusion of the frontal lobe on the right and occipital lobe on the left. This is reversed in cases of situs inversus [42].

Overall, the brain shows multiple anatomical asymmetries. In a study of 171,141 brains scans derived from 99 data sets worldwide, Kong et al. [43] divided the brain into 34 distinct regions, with overall thickness of the cortex larger on the left and overall surface area larger on the right. On both measures, as many regions showed leftward as showed rightward asymmetry, with only a small minority showing no measurable asymmetry. The two measures, though, showed different associations. The frontal regions tended to be thicker on the left while the posterior one tended to be thicker on the eight, a pattern which the authors suggest may derive from the Yakoklevian torque. It was surface area, though, which showed greater association with functional asymmetries. The largest asymmetries in surface area were within language-related areas, including a leftward advantage in a posterior region of Broca's area and the transverse temporal gyrus (part of Wernicke's area), and a rightward advantage in an anterior region of Broca's area. The opposite asymmetries within Broca's area suggest two different circuits involved in language, with the leftward circuit connecting Broca's and Wernicke's areas involved in phonology and syntax. The role of the rightward circuit is not so clear.

Functionally, too, it is becoming increasingly evident that there are several, perhaps many, independent dimensions of laterality. Liu et al. [44] factor analysed laterality indices derived from intrinsic brain activity in the resting brain, revealing four independent factors. Two were left-lateralized, one corresponding to the language network and the other the default-mode network, and the other two were right-lateralized corresponding to a visual network and an attentional one. Badzakova-Trajkov et al. [45] similarly carried out a factor analysis of functional asymmetries while participants undertook language tasks, an attentional task, and a face-recognition task, which yielded three independent factors, a left-lateralized one corresponding to the language network and two right-lateralized networks corresponding to the face-processing network and the attentional network. The right-lateralized face-processing network was largely homologous with the left-lateralized language network, yet uncorrelated with it.

Häberling et al. [46] undertook a further factor analysis of laterality indices while participants performed various left-lateralized tasks, and found three independent factors, representing a language circuit, a gesture-related circuit associated with handedness, and another gesture-related circuit independent of handedness. These finding raised speculation as to how the mirror-neuron system might have lateralized and fissioned into separate subcircuits in the process of hominin evolution.

Orthogonal factor analysis provides a convenient way to identify lateralized networks that are independent of one another and, at least as a first approximation, provide a useful means of determining just how many dimensions of laterality there are.

#### *2.4. Cerebral Asymmetry in Animals*

Evidence for cerebral asymmetries in a wide variety of animals is now abundant (see [47] for review). One general finding is a right-hemisphere dominance for emotion, which seems to be present in all primates so far investigated, including humans [48]. It seems to be true of other animals as well, including dogs [49], horses [50], and birds [51], and probably goes far back in the evolution of vertebrates. Right-hemisphere biases also appear to be unrelated to handedness or motor asymmetries [51]. From an evolutionary perceptive, it may reflect a left-hemispheric disposition to approach and the right hemisphere to avoidance [52].

In humans, the planum temporale overlaps with Wernicke's area, one of the major language areas, and is larger on the left than on the right [53], but the same asymmetry is present in great apes [54–56], and in both adult [57] and infant baboons [58]. This asymmetry may therefore date back at least to the common ancestor of humans, great apes and Old World monkeys, 30–40 million years ago, and is not specifically connected to language.

The other major cortical language area, Broca's area, is more complex. Its anterior portion, area 44 (*pars opercularis*) is part of the language network in humans, and is larger on the left [59] (According to Kong et al. [43] the other portion, area 45 (*pars triangularis*) is larger on the right, while Keller et al. [59] find no asymmetry). Cantalupo and Hopkins [60] report that the homolog of Broca's area in chimpanzees is also larger on the left. Graïc et al. [61] report a structural asymmetry in area 44 of the chimpanzee characterised by smaller neurons, perhaps suggesting increased computational capacity. In this and other respects, the cyto-architectural structure of area 44 seems to resemble closely that in humans.

The emergence of language in humans, though, may be not so much a question of the size of Broca's or Wernicke's areas as of their connectivity. Berwick and Chomsky [39] suggest that two circuits connecting these areas, both present in the chimpanzee, are connected ("a slight rewiring") in the human brain to create a loop that gave us syntax. This occurred, they say, uniquely in humans within the last 100,000 years, "in barely a flick of an eye in evolutionary time" (p. 67). This seems to be more or less pure conjecture. Friederici [62] has suggested similarly but more cautiously that humans evolved a stronger left dorsal connection between these areas than in non-human primates, and that it was this left-sided circuit that enabled the hierarchical structure of language.

From a functional perspective, Friederici's analysis is based on studies showing that humans can detect the hierarchical embedding in sequences of the form (A3(A2(A1B1)B2)B3) (double embedding of this type, when applied to sentences, can be very difficult even for humans to process—an example is *The cat that the dog that the man kicked chased miaowed*), whereas non-human primates cannot [63], and that human processing of such sequences activates area 44. A difficulty with this analysis is that processing sequences of this kind need not involve any understanding of embedding at all; one might simply note that three As are followed by three Bs [64,65]. It is not yet entirely clear how seemingly similar frontotemporal circuits can give rise to language in humans but not in non-human primates, or whether there is indeed a critical difference between apes and humans in this circuitry.

#### *2.5. Cerebellar Asymmetries*

The cerebellum is often neglected in accounts of brain asymmetry, but it too shows functional and structural asymmetries, which tend to mirror asymmetries of the cerebrum; that is, leftward activity accompanies rightward activity in the cortex, and vice versa. In a follow up from the study by Liu et al. [44] of cortical asymmetries in the resting brain, activity on each side of the cerebellum correlated with activity in the association cortex on the opposite side [66]. This implied large-scale circuits combining cerebellum and cortex, with the cerebellum mapping in roughly homotopic fashion onto the association cortex. Cerebellar asymmetry also mirrored cortical asymmetry during a language task, but did not map onto asymmetries of the motor cortex itself. In a similar follow up from the study by Badzakova-Trajkov et al. [45], factor analysis of asymmetrical brain activity induced by language tasks and observations of manual gestures revealed two independent networks, one right lateralized in the cerebellum and left lateralized in the language areas of the brain, and the other associated with handedness and gesture but with no cerebellar involvement [67].

The role of the cerebellum in the hemispheric asymmetry for language gains further support from a recent study showing a correlation between left-hemispheric dominance for perception of dichotically presented syllables, and a rightward asymmetry in the number of voxels in lobule VI of the cerebellum [68]. The dichotic asymmetry also correlated with a leftward asymmetry of the number of voxels in the amygdala, and to a lesser extent with a leftward voxel asymmetry posterior superior temporal cortex. Although dichotic listening provides a less reliable index of functional asymmetry than does brain imaging itself, the results suggest that subcortical areas contribute more to brain asymmetries than is commonly realised. The authors also note that the human cerebellum has a surface area approximately four-fifths of the neocortex, whereas the proportion in the macaque is only about one-third [69]. This invites the speculation that the cerebellum, generally considered to have its primary role in motor coordination, may have expanded in the course of hominin evolution to play a part in the emergence of language.

In chimpanzees, the cerebellum generally follows the pattern of the Yakoklevian torque observed in the human brain [70]. In a sample of chimpanzees studied by Phillips and Hopkins [71] this pattern was reversed, and there was a rightward bias in the volume of the posterior cerebellum in chimpanzees. This was unrelated to handedness as measured in a coordinated manual task. (Curiously, using the same measures, the authors did find that a leftward bias of the posterior cerebellum was associated with right-handedness in capuchins. Unlike chimpanzees, though, capuchins do not appear to show species-wide handedness, nor do they show the Yakoklevian torque.) A subsequent analysis, though, showed an association of this asymmetry with handedness determined from a tool-using task designed to simulate termite fishing [72]. The authors speculate that the asymmetry associated with tool use may have served as the foundation for the emergence of language.

Aside from the question of asymmetry, a recent study reports epigenetic modifications of DNA in the human cerebellum that sets it apart from that in the chimpanzee or macaque, and may suggest a role in the development of language and cognition [73]. GPS methylation at genes known to be involved in neurodevelopment and synaptic plasticity was even more distinctively human in the cerebellum than in the prefrontal cortex. The author suggest that their results "highlight the value of tissue-specific species comparisons of methylation and are consistent with an important role for the cerebellum in human brain evolution.

#### **3. The Genetics of Laterality**

#### *3.1. Handedness*

Historically, attempts to discover the genetic basis of functional laterality have focused largely on handedness, presumably because it is easier to measure than brain asymmetry. Although left-handedness is associated with cultural influences, it is also highly polygenic, as indicated by genome-wide studies of the association between handedness and genetic loci, e.g., [74–76]. These studies clearly rule out single-gene models that have hitherto been popular, e.g., [77,78]. The largest study to date examined individuals from 1,766,671 individuals, combined from the UK Biobank [79] and the International Handedness Consortium, found 41 loci associated with left-handedness, and 7 different loci associated with ambidexterity [80]. A total of 11.9 percent of males were left-handed or ambidextrous, compared with only 9.3 percent of females, a difference comparable to that found in other large-scale studies. Left-handedness was also associated with genetic loci implicated in a number of phenotypical conditions, including schizophrenia, autism, bipolar disorder, neuroticism, mood swings, and educational attainment.

Using an additive model, the authors estimated that genetic effects accounted for 11.9 percent of the variance, shared environment accounted for 4.6 percent, but the largest portion, 83.6 percent, came from individual environmental effects. Dropping shared environment from the model raised the genetic component to 19.7 percent, closer to the 25 percent estimated from twin studies [75,81]. There appears to be still some uncertainty as to how to assess the genetic contribution.

Ambidexterity has often been lumped together with left-handedness, but the two were unrelated genetically. Ambidexterity also showed a different profile of associations with other traits, including a negative genetic correlation with educational attainment. Earlier studies had shown decrements in educational attainment among the ambidextrous relative to left- or right-handers [82,83].

In an overlapping analysis of 501,730 individuals from the UK Biobank, de Kovel et al. [84] revealed that left-handedness was higher in those with lower birthweight, among multiple births, those born in certain seasons of birth, children with lower incidence of breastfeeding, and males, with each of these effects being significant independently of all the others. Others have reported an association of left-handedness with schizophrenia [85], autism [86] and dyslexia [87]. De Kovel et al. refer to a similar analysis based on a large US cohort showing similar association, with the addition of increased left-handedness, among African Americans and those with an older mother [88]. As in the larger study described above, a genome-wide association analysis showed left-handedness to be significantly but only weakly heritable genetically. The bias toward right-handedness, then, may be universal, but subject to variation and possible reversal through extraneous influences, some cultural, some pathological, and some genetic.

This idea of a universal bias is not without precedent. Laland [89,90] suggested that all humans are born with a biological bias to be right-handed, but that deviations result from external pressures. The primary pressure comes from parents, consistent with evidence that the incidence of left-handedness is increased if one parent is left-handed, and more so if both are left-handed. This association has also been taken to support a genetic basis for left-handedness (or the absence of right-handedness), but may equally be due to parental influence. Given the evidence summarised above, though, there are probably additional influences. As a first approximation, then, there may be a universal bias toward right-handedness, but malleable enough to permit variations without undue disadvantage.

Although genetic studies show multiple genetic associations with handedness, these genes may represent different conditions that influence handedness, rather than being intrinsic to handedness itself. An example is the LRRTM1 gene, a maternally suppressed gene associated paternally with handedness and dyslexia; when inherited through the father a particular haplotype consisting of minor alleles at three locations significantly shifted handedness toward the left [91]—a finding partially confirmed elsewhere [92], This same haplotype was over-transmitted paternally in those with schizophrenia. These effects were discovered in dyslexic samples, and were not evident in a Chinese sample or in other samples from the general population, including the large-scale study described above [79].

#### *3.2. Cerebral Asymmetry*

Estimates of cerebral asymmetry based on brain imaging paint a similar picture. In a brain-wide genome-wide analysis in 32,256 individuals, Sha et al. [93] found 41 locations for cerebral asymmetry, parcellated into 34 cortical regions per hemisphere and 7 subcortical regions. Among these, they found 21 distinct, highly significant genomic loci for the different aspects of brain asymmetry. Ten of these were associated with cytoskeletal development, while the remaining 11 were mostly with brain development. These included significant genetic overlaps with autism, schizophrenia, and educational achievement. Earlier studies had shown direct associations of cerebral asymmetry with dyslexia [94], Alzheimer's disease (e.g., [95], ADHD [96], and depression [97]. In all cases, the negative aspects were associated with deviations away from normal asymmetries. Although some of these variables also correlated with handedness in Sha et al.'s study, there was no significant genetic overlap between handedness and structural brain asymmetries, although five individual markers (SNPs) were associated with both. Many of the asymmetries were strong, but their heritabilities were low. As mentioned earlier, situs inversus does not systematically reverse handedness or the normal cerebral asymmetries, with the exception of the Yakoklevian torque.

Again, these findings concur with those based on handedness in suggesting a fundamental but universal bias, with variations imposed by environmental and other conditions, some of possibly genetic origin. Sha et al. conclude from their findings that the development of brain asymmetry is "tightly constrained and largely genetically invariant in the population." The most parsimonious conclusion is that this universal bias also underlies the situs of the internal organs; Brandler and Paracchini [98] suggest that "the mechanisms for establishing LR asymmetry in the body are reused for brain midline development, which in turn influences traits such as handedness and reading ability" (p. 88).

This scenario need not contradict the evidence of relative independence among handedness, different dimensions of cerebral asymmetry, and situs of internal organs. The fundamental asymmetry is invoked where it proves adaptive, even though against the pressure toward symmetry in the bilateria. This is especially true of situs, but less so in handedness and or the various aspects of brain asymmetry where there may be some advantage to maintaining variation—a possibility explored by Ghirlanda and Vallortigara [26]. Evolution itself depends on variation, and within social species such as our own, variations in demeanour, cognition, skill, and personality provide for effective social living, allowing individuals to take multiple specialized roles. Száthmary [99] writes that language, itself strongly lateralized and subject to individual variation, was one of the seven major transitions in evolution, offering something unprecedented—the "negotiated division of labour" (p. 10,109). Whether it was indeed a major transition, or simply a result of progressive evolution is a moot point, and the evolution of complex societies depends not only on language but also on individual differences in other domains as well, including spatial abilities, creativity, athleticism, and computational abilities. We need, or have needed, butchers, bakers, candlestick makers, and software engineers. Genetically, such diversity need not be construed as group selection, but rather as a loosening of genetic determinism.

The universal bias toward asymmetry, then, appears to be most strongly expressed in the situs of internal organs, where deviations from asymmetry are maladaptive. It is also strongly expressed in cerebral asymmetry for language, where deviations may result in language disorders. The bias itself may be universal with deviations only due to extraneous conditions, some pathological, some cultural, and some themselves genetic. For example, a mutation of the FOXP2 gene results in a severe speech impediment, and brain imaging showed that members of an extended family affected by the mutation, unlike their unaffected relatives, showed no activation in Broca's area while covertly generating verbs [100]; the activation seemed to be scattered and to exhibit no consistent asymmetry. Handedness, though, seems to be largely unaffected, with one study showing 12 of the 15 members of the family to be right-handed [101].

The universal bias seems to be less strongly expressed in handedness, where deviations may be adaptive if maintained in a minority. It probably varies across species, but is absent in most animals, where there is no species-wide difference in dominance or preference between left and right forelimbs. That is, the ratio is approximately 0.5, with variations from around equality due only to chance. Laland [89] estimates a bias of 0.78 in humans, so that in the absence of extraneous influences 78 percent of the population would be right-handed, but parental or cultural influences increase it to approximately 90 percent overall. He suggests ratios of 0.8 to 0.9 in Neanderthals, 0.61 in Middle Pleistocene hominins, 0.57 in Lower Pleistocene hominids, and 0.56 in chimpanzees. The bias may be overestimated in Neanderthals, who may have been sufficiently human-like for a cultural influence increasing the overall incidence of right-handedness itself. The bias runs counter to the otherwise general bilateral symmetry of the limbs, and may be largely restricted to bipedal species.

If there is indeed a fundamental bias underling situs as well as handedness and cerebral asymmetries, what is its origin? Morgan and I [102] (readers tempted to consult this article should ignore the Abstract, which was inadvertently substituted from another article) once suggested that it was coded in the oocyte rather than in the genes themselves, and favoured development on the left. It may even depend on the chirality (left–right asymmetry) at the molecular level [103–105]. The asymmetries of the internal organs are governed at the earliest stages by an asymmetry of the cilia, hair-like organelles on the surface of cells, and this directs the asymmetry of a genetic sequence (the Nodal-Lefty-Pitx2 cascade) [106]. Cooke [107] outlines a scenario whereby the asymmetry of the cilia themselves is governed by the alignment of chiral molecules, creating a leftward flow of morphogenes across the embryo, which in turn guides the asymmetrical morphogenes of internal organs through a cascade of genetic influences. These ideas remain speculative,

but imply that asymmetry—or symmetry breaking—is not restricted to humans, or even to vertebrates, but is a fundamental property of living matter.

Whether the asymmetry of the cilia can account for right-handedness, though, remains uncertain. Afzelius and Stenram [108] report on 239 cases of immotile-cilia syndrome, a rare condition in which the cilia are either absent or stationary. In these cases, one might expect random asymmetry, such that 50 percent would have situs inversus and be left-handed. In fact the figures were 44 percent and 14 percent, respectively. This suggest a bias other than that due to ciliary motility, especially in the case of handedness, where the bias was only slightly above the 10–12 percent found in the normal population. Cultural or familial influences may be strong even in the absence of a biological bias.

That said, asymmetries of the hands and brain are clearly more variable than that of situs, where departures from normal asymmetry are often maladaptive. Immotile cilia syndrome, with its high incidence of situs inversus, is accompanied by disorders of the respiratory tract, including sinusitis, rhinitis and bronchitis, and the combination of these with situs inversus is known as Kartagener syndrome, afflicting approximately one in 22,000 [108]. Departures from right-handedness and left-cerebral representation of language are far less drastic, and may even be adaptive in giving rise to special minority talents, as suggested earlier.

This raises the question as to whether disorders associated with lateralization are truly "disorders," or simply part of the fabric of human existence. Dyslexia is often associated with creativity, and even a number of well-known authors, such as Agatha Christie, Gustave Flaubert, and Evelyn Waugh, are said to have been dyslexic. Normal reading depends on an area known as the visual word form area usurping the left side of the occipito-temporal region brain concerned with visual shape analysis. This implies that visual processing can be diminished, or at least altered, when children learn to read [109]. This might explain the special talents of artists, such as Andy Warhol, Pablo Picasso o0 Robert Rauschenberg, who are also said to have been dyslexic. Leonardo da Vinci is often mentioned as another example, although his mirror writing might have been not so much a disability as a disguise. He was, however, left-handed, at least when writing.

Even mental illnesses may be adaptive, or once were so. Kauffman [110] points out that hallucinations were at one time considered normal, and played a part in the lives of visionaries, such as Jesus of Nazareth, St Paul of Tarsus, and even Socrates, and suggests that it was through the writing of Voltaire, Darwin, and Freud that they began to be associated with psychiatric illness. Creativity, too, has long been associated with schizophrenia and bipolar disorders, and research also suggests a genetic link [111]. Nature and culture may have combined to maintain a diversity and creativity of benefit to the species.

#### **4. Conclusions**

The emergence of animals that move created pressure toward bilateral symmetry, and the establishment of the vast clade of animals known as the bilateria. This pressure was due largely to the absence of asymmetrical influences from the natural environment—or what physicists call the conservation of parity. Departures from bilateral symmetry in movement or sensory input could be perilous; Martin Gardner [112] once put it like this:

The slightest loss of bilateral symmetry, such as the loss of a right eye, would have immediate negative value for the survival of any animal. An enemy could sneak up unobserved on the right!

(p. 70).

Nevertheless, bodily asymmetry is ubiquitous, especially in the placement of internal organs. It applies to all chordates and presumably far goes back in evolution. Its fundamental basis may even go back close to the origins of life itself, with the emergence of chiral molecules. At the molecular level, we are steeped in asymmetry.

The brain has largely retained its bilaterian symmetry. Over the course of evolution, though, it has also evolved computational functions not directly constrained by inputs from, or outputs on, the immediate environment. This may include emotion, which seems

to be universally characterised by a bias toward the right hemisphere. Operations on the environment seem more likely to be asymmetrically programmed than are reactions to it, and generally favour the left hemisphere. Examples include throwing, the manufacture and use of tools, and language, whether in the form of speech, gesture, or writing. Again, there may be packaging constraints, with face recognition and perhaps music shifted to the right as compensation for the left-sided representation of language. In the large-scale brain-imaging study by Kong et al. [43], the great majority of the 34 regions examined were asymmetrical one way or the other, yet each region was identifiable on either side, and they were all packaged in such a way as to retain an overall symmetry. Indeed for most of the history of medicine the brain was thought to conform to the law of symmetry.

The genetic orchestration of the asymmetries remains elusive. The most parsimonious solution is that they are ultimately dependent on the same fundamental bias that underlies situs of the bodily organs, but are then expressed by the genetic cascades that create the various specializations, each of which may be expressed or perturbed independently. Even if the various cerebral asymmetries so far identified are not dependent on a single underlying event, they may still hark back to the chirality of biological molecules.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The author declares no conflict of interest.

#### **References**


## *Review* **It Is Not Just in the Genes**

**Martina Manns**

Research Division Experimental and Molecular Psychiatry, Department of Psychiatry, Psychotherapy and Preventive Medicine, LWL University Hospital, Ruhr-University Bochum, 44780 Bochum, Germany; Martina.manns@rub.de; Tel.: +49-234-32-21628; Fax: +49-234-32-14377

**Abstract:** Asymmetries in the functional and structural organization of the nervous system are widespread in the animal kingdom and especially characterize the human brain. Although there is little doubt that asymmetries arise through genetic and nongenetic factors, an overarching model to explain the development of functional lateralization patterns is still lacking. Current genetic psychology collects data on genes relevant to brain lateralizations, while animal research provides information on the cellular mechanisms mediating the effects of not only genetic but also environmental factors. This review combines data from human and animal research (especially on birds) and outlines a multi-level model for asymmetry formation. The relative impact of genetic and nongenetic factors varies between different developmental phases and neuronal structures. The basic lateralized organization of a brain is already established through genetically controlled embryonic events. During ongoing development, hemispheric specialization increases for specific functions and subsystems interact to shape the final functional organization of a brain. In particular, these developmental steps are influenced by environmental experiences, which regulate the fine-tuning of neural networks via processes that are referred to as ontogenetic plasticity. The plastic potential of the nervous system could be decisive for the evolutionary success of lateralized brains.

**Keywords:** avian brain; brain asymmetries; hemispheric lateralization; ontogeny; epigenetic; neuronal plasticity; visual system

**1. The Functional Organization of Brain Asymmetries and Its Development**

"A number of embryonic events make up an integrated overture to the posthatching expression of lateralization" Lesley Rogers [1]

#### *1.1. Lateralization Patterns of Neuronal Systems across the Animal Kingdom*

A fundamental organizational principle of our brain is its asymmetries, which encompass both structural and functional differences between the two hemispheres. This characteristic has led to numerous hypotheses and research projects, which have attempted to elucidate the evolutionary and developmental origins of this specific trait [2,3]. However, lateralization of the brain is not specific to humans, but is present in many species across the animal kingdom. Not only vertebrates, but also many invertebrates, such as flies, bees, octopuses or nematodes, show left–right differences in neural organization and behavior [3–9], which suggests that lateralization is a common feature of metazoan nervous systems [10]. Neuronal asymmetries can be observed in all areas of information processing, including perception, cognition, emotion, homeostatic regulation or motor control and are based on neuroanatomical as well as physiological left–right differences [7,11,12]. Lateralization can be present at the individual level, with left-sided dominance for a certain function in half of a population and right-sided dominance in the other half. In other cases, the direction of a lateralized function within a population is aligned, so that lateralization is present at population level [2,11,13]. Comparative studies indicate that some aspects of functional brain lateralizations share a common evolutionary history [3,7,8,14]. It has been suggested that the vertebrate brain is characterized by specific functional dichotomy, with the left

**Citation:** Manns, M. It Is Not Just in the Genes. *Symmetry* **2021**, *13*, 1815. https://doi.org/10.3390/sym13101815

Academic Editor: Sebastian Ocklenburg

Received: 4 September 2021 Accepted: 23 September 2021 Published: 29 September 2021

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hemisphere more strongly involved in routine and approach behavior, while the right hemisphere dominates detection and response to unexpected, novel and potentially pivotal stimuli [15–17]. For example, several species of fish, amphibians and birds react faster when a predator approaches from the left, indicating that right-hemispheric networks are specialized for the detection of potential dangers, while foraging is controlled by the left hemisphere [3,7,8,18]. The processing of social stimuli, such as faces, is also dominated by the right hemisphere [19] in humans [20], sheep [21,22] and chicks [23]. On the other hand, at least in mammals, communicative signals, such as spoken language in humans [24] or other forms of conspecific vocalizations [25,26], are typically processed within the left hemisphere. A widespread behavioral indicator of hemispheric lateralization is the preferred use of one extremity, which has been documented in a variety of vertebrate and invertebrate species at individual and population levels [5,27–31]. In humans, handedness is the most obvious asymmetry with about 90% of individual preferring to use their right hand for complex manual tasks like fine-tuned object manipulation or writing [32]. Handedness is related to other behavioral asymmetries, like preferential foot use [33] or cradling bias [34], while deviations from the typical pattern are associated with psychiatric or developmental disorders [2,35–37]. Therefore, handedness is used as the favorite measure for correlating functional lateralization with structural left–right differences and genetic variations ([e.g., [12,32,36,38]).

#### *1.2. Understanding Ontogeny of Neuronal Asymmetries—An Unfinished Business*

Despite increasing knowledge about the relationships between different functional lateralizations and their structural foundations, our understanding of the underlying ontogenetic mechanisms is still limited. The presence of population-level lateralizations and cross-species similarities makes it likely that neuronal asymmetries have developed under phylogenetic pressure and, therefore, have a genetic basis [7,8,39,40]. However, human and animal research currently differ in the approaches and methods used in investigating the mechanisms guiding the development of a functional lateralized brain and, therefore, there has only been limited integration of knowledge between research approaches [41]. In some animal models, the genetically controlled events that drive the development of neuronal asymmetries have been studied in detail. In the nervous system of the nematode *Caenorhabditis elegans*, for example, there are pairs of chemoperceptive neurons, which are characterized by molecular left–right differences and different connectivity patterns that are related to their differential functional embedding [42,43]. Molecular genetic studies identified a complex regulation network comprising transcription factors, microRNAs, chromatin regulators and intercellular signals, which determine the asymmetric features of these specific neurons [44]. A second well-studied example is the epithalamus of vertebrate brains, which connects limbic regions of the forebrain with hindbrain motor circuits and which is characterized by evolutionarily conserved asymmetries within the pineal complex and the adjacent habenular nuclei. The molecular pathways that control these asymmetries have mainly been elucidated in studies with larvae of the zebrafish. Here, too, it is a chain of gene expression cascades that underlie the development of lateralization in this area [42,45,46]. Other genes are persistently expressed asymmetrically within the adult forebrain of zebrafish [47].

In human research, however, the first popular models, such as the right shift [48] or dextral chance [49] theory, suggested a single gene origin for human brain lateralization and attributed the left-hemispheric dominance for language processing and hand use to a common genetic factor. Their predictions fit data on the prevalence of handedness and language lateralization, but they did not explain the nature, as well as the action, of such a factor. However, recent meta-analysis studies have shown that the associations between language lateralization and motor asymmetries are much weaker than previously assumed [50]. Currently, research concentrates on the identification of genes that regulate functional and structural lateralization using large-scale heritability and genome-wide association (GWAS), or single nucleotide (SNP) variation studies to find associations between gene variants and

phenotypic lateralizations. These studies have reported an increasing number of genes and their variants related to lateralization pattern. One recent study even identified multifaceted gene networks associated with different aspects of anatomical brain asymmetries [51]. It has also been suggested that the impact of single genes is small and functional lateralizations are polygenic traits [38,52–56]. A recent study, for instance, detected 41 gene loci associated with left-handedness and seven associated with ambidexterity [52]. This also suggests that different manifestations of a trait can be controlled by different types of genes, which are either relevant during different phases of development or which influence discrete differentiation processes of the underlying neural networks.

In general, additive genetic effects account for less than one quarter of the variance in human handedness data, while nonshared environmental factors explain the remaining variance [52,57]. This is not surprising since neuronal systems always differentiate in close interactions with environmental experiences and genes alone do not explain the functional organization of neuronal systems [58]. This implies that the emergence of a functional lateralization pattern can only be understood by elucidating how genes and the environment interact to shape the functional organization of a lateralized brain [2,3,14,59–63]. It must also be considered how noncoding microRNA [64], or epigenetic mechanisms, which affect gene activity and expression by modifying DNA accessibility or chromatin structure, mediate long-term effects of gene–environment interactions [37,63].

Research has reported a potpourri of environmental factors influencing lateralization patterns in humans, including sex hormones [65], stress experience [66], sensory input, learning, birthweight, location and season of birth, breast feeding and cultural constraints [32,53]. These influences underline, on the one hand, the general role of environmental factors, while on the other hand, the lack of specificity of some factors suggests that deviations from genetically controlled patterns simply reflect random stochastic asymmetry [67].

#### *1.3. Structural Foundations of Functional Asymmetries*

Since neuronal functions are based on the organization of specific neuronal networks or cells, it is necessary to clarify how exactly structural and functional asymmetries are related. An increasing number studies have reported structure–function associations, but have also provided an inconsistent pattern. However, it is important to differentiate that left–right differences in the structure of neural circuits can be realized on different organizational levels, from the cellular to the macroscopic level.

An obvious global shape asymmetry of the human brain is the so called "cerebral torque", which refers to a counter-clockwise twist of the whole brain along the anterior– posterior axis [68,69]. At macroscopic level, the left hemisphere has a thicker cortex but a smaller surface area relative to the right hemisphere [70]. Region-specific left–right differences are reported in size and shape [70–72] and connectivity [73,74], as well the cellular and molecular organization [75,76]. Similar cortical asymmetries are also present in chimpanzees [77–79]. The left-hemispheric dominance of language processing is related to left–right differences in the microcircuitry of cortical columns in the posterior part of the superior temporal gyrus [80]. Moreover, there are function-related asymmetries in the hippocampus and subcortical structures in humans [71] and other mammals [81]. Handedness for instance is related to asymmetries within the nigrostriatal dopaminergic system in humans [82] and rodents [83–85].

Cortical left–right differences emerge early during development in humans [86,87], but also in nonhuman primates [88]. The cortical torque can be detected by the second trimester of gestation [68,89], while asymmetry of perisylvian language-related cortical regions appears during the third trimester [90–92]. Motor asymmetries can be observed even earlier. Human fetuses tend to make more movements with their right arms and preferentially suck the right thumb from the 12th gestational week onwards [93]. These motor asymmetries are related to postnatal handedness [94]. In relation to this behavioral lateralization, the fetal spinal cord segments innervating hands and arms display asymmetries in gene expression and DNA methylation at the end of the first trimester [95].

In sum, average left–right differences of global brain anatomy, which emerge early during development, suggest a developmental program that is genetically determined [96]. However, when analyzing specific cognitive functions, gene–structure interrelations are less detectable. Twin studies, for instance, indicate that pre- and postnatal events can affect asymmetry during development of the planum temporal [97,98]. Accordingly, a recent large-scale study did not find significant associations between cortical asymmetries and language lateralization [99]. There is also no significant relation between cortical asymmetries and handedness [70]. The lack of correlations may not come as a surprise since the macroscopic cortical features do not necessarily represent the internal microscopic organization. It is conceivable that functional asymmetries only emerge on the cellular, synaptic or neurophysiological level. This means that it is necessary to understand how neuronal asymmetries arise at precisely this cellular level. To this end, findings from developmental neurosciences have to be integrated into models of asymmetry formation. Experiments with animal models have shown that activity-dependent processes triggered by internal or external signals are decisive for the functional maturation of neural networks [100–103]. In the following, I will, therefore, first summarize what is known about the role of genetic factors for asymmetry formation during different developmental phases. I then illustrate the possible effects of environmental factors as suggested by the light-dependent development of visual asymmetries in birds.

#### **2. Potential Roles of Genetic Factors for Asymmetry Formation**

The relative importance of genes and the environment depends on the species examined, the specific neuronal function and their developmental trajectories, as well as the level of analysis [61]. This means that we have to differentiate the action of gene–environment interactions depending on the development phase. The development of the nervous system can be roughly divided into three phases, during which the degree of hemispheric specialization increases (Figure 1). The first phase comprises the earliest embryological steps, in which the axes of the body plan are determined. The second phase includes the differentiation of neural systems and networks, while processes mediating the refinement of neural connections dominate the third phase. During these phases, different cellular processes dominate development and genes can influence the action of epigenetic factors in different ways, which affect the developing organism (Figure 1):

**Figure 1.** Model of the hierarchical development of brain lateralization—the three main phases of neuronal development are dominated by different cellular processes, which lead to an increasingly lateralized functional organization of the two hemispheres (indicated by the green and orange triangles). In each phase, certain types of genes regulate differentiation and, thus, asymmetry formation.


In the meantime, a number of genes have been discovered that mediate at least one of these actions during asymmetry formation:

#### *2.1. Embryonic Patterning*

Asymmetry formation within neuronal systems starts with breaking the symmetry of the body plan during early embryogenesis in all bilaterian animals, when the primary axes and tissue layers form. Complex cascades of genetic and epigenetic interactions lead to an asymmetrical placement of internal organs, but also induce asymmetries of paired organs like the lungs or the nervous system [104–109]. Determination of the left–right body axis is coordinated by a midline structure called the node. In several species, including humans, symmetry is broken by the rotation of motile cilia, which generate a directed flow that acts as a signal for the asymmetrical expression of a gene cascade, the Nodal signaling pathway. This pathway is remarkably conserved within bilaterian evolution [10,106,108].

This implies that asymmetry formation of body and brain starts with the action of cilia and, therefore, genes controlling generation and motility of cilia could play an early role in the development of neuronal asymmetries [38,110]. Some studies have actually provided evidence for the involvement of cilial genes for handedness—however, only in specific humans populations [38,54,111].

A second critical mechanism during this early phase is the lateralized action of the Nodal pathway. One key player in this signaling cascade is PCSK6, which cleaves the Nodal protoprotein into its biologically active form [10,110,112]. *PCSK6* polymorphism has been associated with human handedness [38,113], but also with structural asymmetries in temporal cortical areas, indicating a potential role of *PCSK6* not only for motoric but also language networks [114].

However, when symmetry breaking processes of visceral and neuronal structures share the same developmental route, one should assume that individuals with reversed visceral organization also display reversed brain asymmetries. A test case involves individuals with situs inversus, where the visceral organs are organized as a mirror image of the default organ position. Situs inversus can occur in, but does not depend on, ciliary dyskinesia [115]. While the typical gross morphological asymmetry of the human brain–cerebral torque is actually reversed in situs inversus, functional and cortical lateralizations are not [115–119], although atypical functional segregation can be more frequent in participants with visceral reversal [115,120]. Similarly, in less complex animals, such as the nematode *C. elegans*, motor lateralization is independent from left–right body asymmetry [121] and zebrafish with situs inversus develop reversed lateralization of some but not all structural and behavioral lateralizations [122]. This suggests that early embryonic patterning processes regulate, to some degree, the establishment of basic brain asymmetries, but lateralization of specific functional modules are presumably shaped by specific cellular mechanisms later during development [119,123].

#### *2.2. Regionalization of Neuronal Substrate*

When the neuronal anlage starts to differentiate region-specific differences, genes playing a role in symmetry breaking of the embryo are also involved in the generation of specific brain asymmetries. The best known example is the Nodal pathway, whereby asymmetrical left-sided Nodal signaling within the developing dorsal diencephalon is required for determining the direction of epithalamic asymmetries [42,45,112,124]. It is conceivable that laterality signals result in asymmetrical expression of neuron-typespecific gene batteries, which are responsible for cell-type-specific structural and functional properties [125].

#### *2.3. Differential Developmental Dynamics*

One consequence of the early left–right patterning is that the left and right hemispheres develop at different speeds. In human embryos, the right hemisphere tends to develop a little earlier than the left one [86] and the lateralized gradient of brain development might contribute to the development of the cerebral torque [69]. Differences in developmental speed of cortical subareas are indicated by specific lateralized gene expression profiles from the fifth week postconception onwards [126]. The early appearance of asymmetrical arm movements in human fetuses can be explained by left–right differences in the differentiation of spinal neurons, since the cortex and spinal cord are not connected at this age [127,128]. As a result of the asymmetrical developmental gradients of the two hemispheres, it is possible that a nongenetic factor, which acts on the developing organism at a certain point in time, differentially influences left- and right-hemispherical neuronal structures. There is, for instance, some evidence that the right hemisphere of human fetuses is generally less subject to external influences than the left one [86].

#### *2.4. Differentiation of Hemisphere-Specific Neuronal Elements*

When the nervous tissue starts to differentiate region-specific neurons and connections, specific genes regulate proliferation, migration and growth of axonal and/or dendritic fibers. Therefore, asymmetrical expression of these genes can account for the asymmetrical differentiation of specific brain regions.

Sun et al. [129,130] identified a couple of genes in perisylvian regions of the human cortex, which are asymmetrically expressed at the end of the first trimester and, therefore, before a neuroanatomical asymmetry of this area can be detected [90,91]. Intriguingly, most of these asymmetrically expressed genes function in signal transduction and gene expression regulation [129,130].

One of these genes is the transcription factor *LMO4*, which is consistently more highly expressed in the right perisylvian cortex of 12–16-week human fetuses and, hence, during a period of high proliferation and migration rate [129,130]. *LMO4* displays higher expression level also in the right forebrain of zebrafish [47], while in the mouse cortex, *LMO4* expression is not constantly lateralized to one side [129,130]. Expression of *LMO4* is confined to postmitotic neurons [131] and regulates key aspects of neuronal differentiation, radial migration of newborn nerve cells and acquisition of neuronal identities [132,133].

Another example is the transcription factor forkhead box P2 gene *FOXP2*, which is involved in neural development and, in particular, in regulating neurogenesis of the embryonal cortex. It is expressed in distinct brain areas from gestational week six onwards and is related to speech development [134]. Intriguingly, *FOXP2* polymorphism is associated with the interindividual variability in hemispheric asymmetries for speech perception [135].

#### *2.5. Ontogenetic Plasticity*

After the establishment of the basic brain organization, neuronal networks typically sharpen their functional efficiency. Growth, stabilization or reduction of synaptic contacts or cell death occur in an activity-dependent manner and are triggered by sensory experience [100,136]. This critical period is likely to amplify expression of genes and proteins that mediate synaptic plasticity. Accordingly, genes that are involved in regulating ontoge-

netic plasticity can affect the asymmetrical development during specific sensitive phases. Asymmetrical expression of these genes can result in a differential sensitivity of left- and right-hemispheric circuits towards stimulation. Karlebach and Francks [137], for instance, identified several asymmetrically expressed genes in the human cortex that are likely to fine-tune electrophysiological and neurotransmission properties of cortical circuits during different phases of development. Additionally, in the rat hippocampus, a dynamic pattern of asymmetrically expressed genes has been identified during the first postnatal weeks, with a large percentage of genes being associated with synaptic function [138]. One example could be the transmembrane molecule LRRTM1 (leucine-rich repeat transmembrane neuronal 1). It interacts at synapses with the extracellular matrix as a regulator of neuronal plasticity [139]. Gene variations have been associated with handedness [53,140,141].

Crucial mediators of ontogenetic plasticity are neurotrophic factors like BDNF (brainderived neurotrophic factor), which mediates activity-dependent synaptic stabilization, axo-dendritic growth, arborization and cell survival [142,143]. It is, therefore, intriguing that BDNF is asymmetrically expressed in the hippocampus of rats, specifically during the first two weeks after birth when neurogenesis rate is high [144]. BDNF might also mediate stress effects in the brain and could, therefore, regulate the well-known action of stress hormones onto brain lateralization [145].

To sum it up, neuronal development is controlled at very different levels of differentiation by genes that are either asymmetrically expressed or whose variants are associated with specific phenotypes. The same function (e.g., handedness) can, therefore, be regulated during different developmental phases by different types of genes. Asymmetrical expression of single genes can be confined to specific developmental phases, while other genes are lateralized up until adulthood. At all levels, nongenetic factors can modulate genetic effect and thereby change the direction and/or degree of lateralization. However, little is yet understood about the neuronal processes through which environmental factors can influence the differentiation of the complex functional organization of lateralized brains. One of the few models in which the influence of a specific environmental factor has been examined in more detail is the visual system of birds. Research on chicks and pigeons has delineated a chain of events that begins with asymmetrical photic stimulation of the embryo in the egg and ends in a lateralized organization of visual processing and cognition [1–3,14,40,59–61,146,147]. This model suggests critical steps for the formation of asymmetries that can serve as a blueprint for a better understanding of the ontogenesis of brain asymmetries in general. These developmental steps are summarized below (Figure 2) and are complemented by findings in other species, especially in humans.

**Figure 2.** The developing visual systems of chicks and pigeons exemplifies how one environmental factor—in this case light—affects the development of brain asymmetries during the three main phases of neuronal development (see text for details).

#### **3. The Avian Visual System as a Model for Ontogenetic Plasticity**

The visual system of chicks and pigeons is lateralized with a pattern that is similar to the lateralization of the human brain. The left hemisphere dominates the discrimination of small optic details, rule learning, categorization and visuomotor control [59–61,147–149]. The right hemisphere on the contrary, is in charge of spatial attention [150] and aspects of social cognition [23]. These hemispherical specializations can be identified very easily by temporarily occluding one eye with an opaque cap. Since the optic nerves cross virtually completely in birds, information from the left eye is primarily directed to the right hemisphere and vice versa. A comparison of monocular and binocular testing, therefore, enables the investigation of hemispherical differences in performances or analysis strategies. Behavioral asymmetries are accompanied by anatomical left–right differences within the ascending visual pathways. In both pigeons and chicks, for example, differences in the projection strength between the two hemispheres can be observed. Major aspects of these asymmetries develop in response to asymmetrical visual stimulation during development. Therefore, light deprivation before and after hatching prevents or modifies visual lateralizations. The comparison of structural and behavioral lateralizations of light-exposed or light-deprived birds makes it possible to unravel critical neuronal processes that mediate light-dependent development (Figure 2) [1–3,14,40,59–61,146,147,151,152].

#### *3.1. Mechanisms during Embryonic Patterning (Phase I)*

As in all vertebrates, asymmetry formation in birds starts during embryonic body patterning [153,154], whereby symmetry breaking is independent from motile cilia [105,106]. At this point of development, light cannot directly affect visual lateralization patterns but there are at least three routes serving as starting points for the induction of asymmetries in the visual system:


from week 38 onwards [169]. During this time, human fetuses are already responsive to sensory stimulation. They are able to memorize auditory stimuli from the external world by the last trimester of pregnancy, with a particular sensitivity to melody contour in both music and language [170,171]. Differential auditory input to the left and right ear because of postural asymmetries, therefore, might affect the development of language lateralizations [172–174].

3. Although visual systems are not developed, there is some evidence that during this phase, light stimulation already affects the establishment of some aspects of lateralization in both chickens and zebrafish [166,175–177] (Figure 2). Transduction mechanisms mediating these light effects are unknown but might include epigenetic mechanisms [166,177]. It is also possible that some genes unfold their actions only after photostimulation [178].

#### *3.2. Mechanisms during Neuronal Differentiation (Phase II)*

It is well known that the differentiation of visual networks is critically influenced by visual stimulation (e.g., [100]], and it is therefore not surprising that unbalanced light stimulation differentially affects left- and right-hemispheric developmental processes during species-specific sensitive phases [61,179]. Some behaviors and anatomical asymmetries only develop after embryonic light stimulation [180–182] and can be reversed by altered visual experience before (chicks, [183]) or after (pigeons [184,185]) hatching. In chicks, the outgrowth of visual fibers is influenced by light stimulation, resulting in a transiently stronger innervation of the right visual forebrain. Thereby, the action of light is modified by corticosterone, testosterone and estradiol [1,14,146,147,149,186]. The modulatory action of steroid hormones is in line with the often described sex- and stress-effects on human and nonhuman lateralization patterns [66,187,188]. In pigeons, left–right differences in cell size and projection strength differentiate in response to asymmetric photic stimulation [180,182,184,185,189]. Posthatch experimental manipulations have shown that starting with asymmetrical retinal activity [190], asymmetrical differentiation within the ascending visual system is mediated partly by BDNF-dependent processes [191,192].

The avian models exemplify how an environmental factor shapes the generation of neuronal asymmetries by modifying specific bottom-up systems. In a similar way, left–right differences in spectrotemporal selectivity of neurons in the auditory cortex of mice develop depending on hearing experience, which is related to the left-hemispheric dominance for the analysis of vocalization features [193]. In humans, visual experience can affect handedness [173], head turning preference [194] or lateralized face-processing competence [195,196].

However, an asymmetrical sensory trigger, such as light, not only enhances differentiation of the stronger stimulated hemisphere but also modifies the balance of left- and right-hemispheric development. A detailed analysis of light- and dark-incubated pigeons, for instance, revealed that light induces a left-hemispheric increase in visuoperceptual skills but simultaneously decreases visuomotor speed within the right hemisphere [182]. At the neuroanatomical level, embryonic light stimulation does not increase the bilateral innervation of the more strongly stimulated left brain side, but rather decreases input to the right side [180].

Presumably, interdependent left- and right-hemispheric developmental processes also play a role in the experience-dependent specialization of the human cortex, as indicated by the distribution of hemispheric language and face recognition processing. While the visual word form area in the left hemisphere becomes specialized while learning to read, the right hemisphere develops face recognition dominance. This suggests that the hemispheric organization of face recognition and of word recognition does not develop independently, and that word lateralization may precede and drive later face lateralization [196,197].

#### *3.3. Consolidation of Functional Asymmetries (Phase III)*

The ontogeny of visual asymmetries in birds is profoundly triggered within the developing ascending visual pathways but cognitive asymmetries emerge only at a higher (forebrain) processing level [60,148]. This means that asymmetries, which are induced within bottom-up systems, have to be transferred onto higher brain structures. At this level, they might interact with inherent or light-independent asymmetries (see above) and thereby sculpt and stabilize the final functional organization of the visual brain. In the pigeon, these processes mainly take place after hatching, when light input is normally symmetrical. During this phase, lateralization can still be modified by manipulating the visual experience [59,60,152,184,190]. It is likely that top-down as well as commissural mechanisms play a critical role in these stabilization processes [59,60,185,189,198,199]. As a consequence, relevant top-down and/or commissural systems develop their own asymmetrical properties for controlling asymmetrical decision-making and behaviors, but also for determining the degree of interhemispheric crosstalk. For example, left-hemispheric dominance for conflict choices is related to the asymmetrical action of top-down projections from the forebrain [198]. Light-dependent efficiency of interhemispheric integration has been shown in chicks, where only light-stimulated individuals can efficiently allocate food searching to the left and predator vigilance to the right hemisphere [200]. Also, only light-exposed chicks can use object (left-hemisphere)- as well as position (right-hemispheric)-dependent cues in food searching tasks [201,202]. A study with pigeons showed that only light-stimulated birds integrate hemispheric-specific knowledge for solving a task that cannot be correctly answered with information of one hemisphere alone [199]. Relevance of interhemispheric mechanisms for the generation and modulation of hemispheric-specific functions is in line with studies exploring the role of the corpus callosum for brain lateralizations [203,204]. The avian model suggests that top-down and commissural systems unfold their effects mainly at the end of asymmetry formation and modulate the interaction of more or less strongly lateralized neuronal networks in the left and right hemispheres [148]. To this regard, these processes shape the final functional organization of lateralized cognitive modules.

#### **4. Conclusions**

Studies on the genetic basis and/or environmental influences on the formation of asymmetries in humans and other animals have shown that the development of a lateralized functional architecture of the brain is to be understood as an example of ontogenetic plasticity. Genes and environmental factors play different but intertwined and complementary roles that can be specific to certain processing modules. The final functional lateralization pattern is then the result of hierarchical processes that build on one another. Genetically controlled early embryonic developmental steps set the framework for hemispherical differences and can be indicated by gross morphological asymmetries in volume and/ or shape of gray and white matter. Epigenetic processes lead to increasing hemispherical specialization and control dynamics of interhemispheric communication. This means that no factor alone can explain the variance of lateralization patterns in a population; it is the sum of individual experiences, which shape individual brain lateralization. It is possible to identify general roles of single genes or environmental factors, but only their interplay within a specific environment determines the functional outcome. Consequently, single factors can only explain limited variance in the lateralization pattern within a population.

This flexibility enables fluctuating lateralization patterns within a population depending on the ecological requirements. Recent field studies showed, for instance, that factors such as predator pressure, environmental pollutants or seasonal conditions can modify brain asymmetries [205–207]. Humans have cultural constraints affecting, for example, the prevalence of left-handedness [208]. It is conceivable that the specific ecological or social conditions account for population-level lateralization in humans, which is absent in other animals species [208]. Ontogenetic plasticity, however, can be a general mechanism that enhances the evolutionary benefit of brain asymmetries [61,208].

**Funding:** This work was funded by the DFG (grant number MA4485/2-2).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The author declares no conflict of interest.

#### **References**

