Compared to the almost infinite complexity of vertebrate cognition and behavior, the number of genes influencing central nervous system development is staggeringly small (Kadakkuzha and Puthanveettil, 2013). Thus, understanding the molecular mechanism underlying the epigenetics of vertebrate central nervous system architecture has become an issue central to neuroscience (Kundakovic and Champagne, 2015).

One fundamental principle of brain organization is lateralization, i.e. structural or functional difference between the left and the right hemisphere of the brain (Corballis, 2014). Lateralization is a conserved feature across the vertebrate lineage (Ströckens et al., 2013; Ocklenburg et al., 2013a; Bisazza et al., 1998; Rogers et al., 2012; Versace and Vallortigara, 2015) and recent studies strongly suggest it is also present in invertebrates (Frasnelli et al., 2012). This ubiquity of behavioral and brain lateralization strongly supports the idea that lateralized central nervous system organization provides an evolutionary advantage (Vallortigara and Rogers, 2005). Suggestions about why a lateralized brain would increase an organism’s fitness include avoidance of unnecessary duplication of neuronal activity in both hemispheres, faster neuronal processing due to not being constrained by slow callosal transfer of information between the hemispheres and better coordination of unilateral behaviors in swarms or other social groups of animals (Vallortigara and Rogers, 2005; Corballis, 2009). In humans, hemispheric asymmetries have been shown in almost all major cognitive systems (Ocklenburg et al., 2014a) including language (Friederici, 2011; Sepeta et al., 2016), memory (Giammattei and Arndt, 2012; Tat and Azuma, 2016; Habib et al., 2003), attention (Falasca et al., 2015; Duecker et al., 2013), emotional processing (Demaree et al., 2005), face perception (De Winter et al., 2015), working memory (Langel et al., 2014; Nagel et al., 2013) and executive functions (Ocklenburg et al., 2011a, 2012; Stock and Beste, 2014).

By far the most widely investigated manifestation of lateralization in humans is handedness (Corballis, 2014). Importantly, handedness is related to the lateralized organization of cognitive systems in the human brain (Ocklenburg et al., 2014b; Frässle et al., 2016). For example, left-hemispheric language dominance is found in 96% of right-handed subjects, but only in 73% of left-handed subjects (Knecht et al., 2000). The relevance of handedness has recently been highlighted by Willems et al. (Willems et al., 2014), who state that it is one of the most important factors influencing the individual brain organization and that explicit inclusion of left-handers in experimental studies has strongly improved our understanding of language, motor behavior and visual processing. Handedness might not only be a behavioral proxy for individual brain organization, but is also interesting from a clinical perspective: A variety of neuropsychiatric and developmental disorders like autism spectrum disorders (Colby and Parkison, 1977; Forrester et al., 2014; Preslar et al., 2014; Rysstad and Pedersen, 2016), depression (Denny, 2009; Elias et al., 2001; Logue et al., 2015), bipolar disorder (van Dyck et al., 2012; Nowakowska et al., 2008), anxiety disorders (Logue et al., 2015; Hicks and Pellegrini, 1978; Orme, 1970; Wright and Hardie, 2012; Hardie et al., 2016; Lyle et al., 2013), schizophrenia (Hirnstein and Hugdahl, 2014; Dragovic and Hammond, 2005; Sommer et al., 2001) or alcoholism (Denny, 2011; Mandal et al., 2000; Sperling et al., 2000) has been associated with left- and mixed-handedness. Thus, understanding the ontogenesis of handedness and hemispheric asymmetries in general could potentially yield important insights into pathogenesis of these disorders.

However, despite their importance for many aspects of brain organization, the ontogenetic background of brain asymmetries is still far from being understood. Initially, single gene theories have been suggested to explain the emergence of handedness as a function of one gene with two alleles (Annett, 1998; McManus, 1985). However, recent genome wide association studies failed to detect any genome-wide significant single nucleotide polymorphisms, refuting single gene theories (Armour et al., 2014; Eriksson et al., 2010). Candidate gene studies revealed a number of genes that display an association with handedness, among them leucine rich repeat transmembrane neuronal 1 (LRRTM1) (Francks et al., 2007), proprotein convertase subtilisin/kexin type 6 (PCSK6) (Scerri et al., 2011; Arning et al., 2013; Brandler et al., 2013) and the androgen receptor gene (AR) (Arning et al., 2015; Hampson and Sankar, 2012; Medland et al., 2005). However, these genes explain only a fraction of the variance in handedness data. Moreover, a number of studies has suggested that only about one quarter of the variance in handedness is attributed to genetic variation, whereas the remaining 75% of variance are explained by non-shared environmental factors (Medland et al., 2006, 2009; Vuoksimaa et al., 2009). These findings highlight the importance of integrating both genetic variation and epigenetic processes modulating gene expression when investigating the ontogenesis of hemispheric asymmetries (Geschwind and Miller, 2001).

Asymmetric gene expression in the fetal cortex has been suggested as the molecular basis of left-right differences in hand-use: Sun et al. (Sun et al., 2005) compared gene expression levels in the right and left perisylvian cortex of the human fetus. At 12 gestational weeks, the authors identified 27 consistently asymmetrically expressed genes, which are mostly responsible for gene expression regulation, signal transduction, and cortical development. One of the consistently asymmetrically expressed genes was LIM Domain Only 4 (LMO4). Further investigation revealed that unilateral variation of Lmo4 expression in embryonic mice suppresses neurogenesis in one hemisphere, leading to the asymmetric functional area formation, neuronal production and axonal projection as well as altered paw preference (Li et al., 2013). Analysis of gene expression in the adult human brain yielded less clear results, since two independent studies found no differences in gene expression between analogous regions across the cerebral hemispheres (Hawrylycz et al., 2012; Pletikos et al., 2014). In a recent study, Karlebach and Francks reanalyzed both datasets and showed that subtle lateralization at single gene level translates to stronger asymmetries at the level of functional gene ontology (GO) groups. The authors found lateralized gene sets to be associated with neuronal electrophysiology, synaptic transmission, nervous system development, and glutamate receptor activity (Karlebach and Francks, 2015).

However, recent research indicates that cortical tissue might not be the optimal choice to investigate the relation of gene expression asymmetries and behavioral asymmetries. Ontogenetically, handedness starts early in development since coordinated hand movements begin 8 weeks post conception (PC), i.e. 10 weeks gestational age, when 85% of fetuses exhibit more right arm than left arm movements (Hepper et al., 1998; de Vries et al., 1985). Investigation of thumb sucking in 274 fetuses revealed that at 13 weeks PC 90% prefer to suck their right thumb whereas only 10% suck their left thumb more often (Hepper et al., 1990, 1991). Interestingly, a follow up study of 75 infants revealed that thumb sucking preference is significantly positively correlated with subsequent handedness: The 60 children showing a right thumb preference were right-handed whereas out of the 15 children displaying a left thumb preference, five were right-handed and 10 were left-handed (Hepper et al., 2005). Importantly, the motor cortex is not yet functionally linked to the spinal cord at that stage of development as the outgrowth of corticospinal projections does not enter the anterior spinal cord before 15 weeks PC (ten Donkelaar et al., 2004). This implies that handedness is unlikely to be under brain control (Hepper et al., 1991) and asymmetrical hand movements have to arise from spinal activity patterns. Thus, it is likely that spinal rather than cortical gene expression asymmetries represent the molecular basis of handedness.

Asymmetrical gene expression patterns are likely to be influenced by epigenetic variation. The most important epigenetic mechanism is DNA methylation. Binding of methyl (-CH 3 ) groups to CpG sites or islands causes a reduction or prevention of transcription and thus gene expression. DNA methylation is confirmed to be involved in the development of basic central nervous system functions like synaptic function, neuronal plasticity, learning and memory (Nikolova and Hariri, 2015; Day et al., 2015; Roth, 2012). Especially intrauterine stressors have been shown to influence DNA methylation (Turecki and Meaney, 2016; Vaiserman, 2015), which is particularly interesting in the context of handedness ontogenesis. Moreover, a recent study showed that methylation plays a role in the ontogenesis of handedness: methylation levels in a CpG block in the promoter region of LRRTM1 were associated with atypical handedness (Leach et al., 2014).

Post-transcriptionally, gene expression is further regulated by microRNAs (miRNAs) that are composed of small, 21–25 nucleotide, non-coding RNAs. In humans and other mammals, miRNAs primarily cause destabilization of target mRNAs instead of reduced translation (Guo et al., 2010). This has also been shown to be relevant for hemispheric asymmetries, as neuronal asymmetries in the nematode Caenorhabditis elegans are controlled for by different miRNAs (Alqadah et al., 2013; Cochella and Hobert, 2012; Johnston and Hobert, 2003; Hsieh et al., 2012).

Pronounced changes in spatiotemporal expression profiles are a key feature of human embryogenesis (Yi et al., 2010) and formation of functional asymmetries in vertebrates has been shown to strongly depend on critical periods in early development (Le Grand et al., 2003; Zappia and Rogers, 1983). To investigate the molecular determinants of human behavioral asymmetries we analyzed asymmetries in genome-wide mRNA expression, miRNA expression and DNA methylation patterns in human fetal spinal cord tissue. Importantly, we specifically wanted to investigate the spinal cord segments innervating arms and hands. While rostral cervical segments (C2–C5) innervate the head, neck and shoulder region, the subsequent segments directly innervate arms and hands with C6 innervating the thumb, C7 innervating the middle finger and C8 innervating the little finger. T1 innervates the medial site of the antecubital fossa (Maynard et al., 1997). Based on the findings on the start of left-right asymmetries in coordinated hand movements (Hepper et al., 1998; de Vries et al., 1985), we focused on fetal tissue samples obtained between 8 and 12 weeks PC.

We hypothesized that gene expression asymmetries between the left and right spinal cord start at 8 weeks PC, as this is the starting point of coordinated asymmetrical hand movements. Based on the findings about the role of non-genetic influence factors for handedness development, we also assumed a pronounced modulation of these mRNA expression asymmetries by asymmetric DNA methylation and asymmetric miRNA expression.