Cortex

Tao Sun

A striking asymmetrical brain function in humans is preferential handedness, with more than 90% of the population more skillful in using the right hand, which is controlled by the left hemisphere. The preferential right-hand use is even detectable during fetal development. Some more sophisticated cognitive functions are also pref- erentially localized in the left or right hemisphere; for example, the left hemisphere is dominant for language and the right hemisphere excels in spatial recognition.

Moreover, anatomical asymmetries, for instance, the sylvian fi ssures and the planum temporale, have been mapped in human brains using imaging techniques such as magnetic resonance imaging (MRI).

In the past decade, using genetic tools and animal models, researchers have dem- onstrated causal relationships between genes and behaviors. Whether brain anatomi- cal and functional asymmetry is regulated by genetic programs is unclear. Even though genetic models of human handedness have been proposed, the “ genes ” that may control preferential hand use in humans have not been identifi ed. Taking advantage of large-throughput genetic screening approaches, we are beginning to uncover the differential gene expression in human left and right hemispheres. These candidate genes can serve as references in revealing the molecular mechanisms of brain asym- metry and handedness in humans and animal models.

Genetic Patterning of the Cerebral Cortex

During the development of the central nervous system (CNS), the forebrain is orga- nized into a three-dimensional structure with anterior – posterior (A-P), dorsal – ventral (D-V), and left – right (L-R) features ( Grove & Fukuchi-Shimogori, 2003 ; Levitt &

Eagleson, 2000 ; O ’ Leary, Chou, & Sahara, 2007 ; O ’ Leary & Nakagawa, 2002 ; Rakic, 1988 ; Sun & Walsh, 2006 ; Sur & Rubenstein, 2005 ). Our knowledge of early brain patterning is largely from investigations of rodent brain structures and functions using genetic tools. Brain patterning is initiated by molecules secreted from patterning centers. At least three patterning centers have been identifi ed: (1) The ventral features

are regulated by Sonic hedgehog (Shh) secreted from the mesendoderm beneath the forebrain ( Chiang et al., 1996 ; Machold et al., 2003 ), (2) the anterior features are controlled by the fi broblast growth factor 8 (Fgf8) expressed in the anterior midline of the forebrain ( Fukuchi-Shimogori & Grove, 2001 ; Garel, Huffman, & Rubenstein, 2003 ), and (3) the dorsal features are regulated by bone morphogenetic proteins (BMPs) and Wnts expressed in the dorsal midline in the cortex ( Bulchand, Grove, Porter, & Tole, 2001 ; Golden et al., 1999 ; Gunhaga et al., 2003 ; H é bert, Mishina, &

McConnell, 2002 ; Monuki, Porter, & Walsh, 2001 ; Zhao et al., 2005 ). Recently, another potential patterning center, the cortical antihem, was identifi ed at the lateral cortical fi eld ( Assimacopoulos, Grove, & Ragsdale, 2003 ).

Along the A-P axis in the cortex, molecules secreted from patterning centers induce downstream gene expression, such as transcription factors. These transcription factors normally have gradient expression; for example, Pax6 is expressed in an anterior-high and posterior-low gradient, while Emx2 and an orphan nuclear receptor chicken oval- bumin upstream promoter-transcription factor 1 (COUP-TF1) are expressed in an anterior-low and posterior-high gradient ( Armentano et al., 2007 ; Bishop, Rubenstein, & O ’ Leary, 2002 ; Liu, Dwyer, & O ’ Leary, 2000 ; Zhou, Tsai, & Tsai, 2001 ). Even though it is unclear how gradient expression of transcription factors is established, the proper expression pattern of these transcription factors is essential for organizing distinct cortical functional areas. Mutation in Pax6 produces expansion of posterior cortical region, resulting in smaller motor cortex in the anterior ( Bishop, Goudreau, & O ’ Leary, 2000 ; Muzio et al., 2002 ). Altering cortical expression levels of Emx2 alone causes a larger or smaller visual cortex in the posterior ( Leing ä rtner et al., 2007 ). As a result, mice with altered Emx2 cortical expression display impaired sensory detection and motor coordination ( Leing ä rtner et al., 2007 ). These results indicate that the proper anatomical formation and functional performance of mouse brains are controlled by precise gene expression and regulation during development.

The organization of functional areas in human brains is more complex than in the mice. Based on conserved functions between human and mouse genes, similar patterning mechanisms in mouse brains may nevertheless apply to humans.

Differential Gene Transcription and Human Brain Asymmetry

If the anterior and posterior organization of functional areas in developing brains is regulated by genetic programs, does the establishment of asymmetrical structures and distinct functions between the left and right hemispheres also have a molecular base?

The molecular regulation of the left – right asymmetry of visceral organs of verte- brate bodies, such as the heart, lungs, and intestines, has been thoroughly studied ( Levin, 2005 ; Wright, 2001 ). Signaling pathways, for instance, Shh, Fgf8, and Nodal

and their downstream genes, have been shown to play a critical role in left – right body determination ( Capdevila, Vogan, Tabin, & Izpisua Belmonte, 2000 ; Hamada, Meno, Watanabe, & Saijoh, 2002 ). An interesting question is whether genes involved in visceral organ asymmetry have differential expression between human left – right hemispheres. If they are differentially expressed in human brains, they likely play a conserved role in regulating brain asymmetry as in controlling visceral organ asym- metry. In a preliminary study, expression patterns of candidate genes for body asym- metry, such as Pitx2 and Wnt molecules, have been tested in 17- to 22-week-old human fetal brains ( Geschwind & Miller, 2001 ). However, no signifi cant differences in gene expression have been observed between the two hemispheres.

A recent study directly measured gene expression levels in the left and right hemi- spheres of human fetal brains using a genetic screening approach ( Sun et al., 2005 ).

In this study, levels of gene transcripts were quantifi ed using the total RNA extracted from the perisylvian regions, the presumptive language center, of human fetal brains at 12, 14, and 19 weeks. To compare expression levels of almost all transcripts either with high or low abundance without bias, gene expression libraries were created using a technique called serial analysis of gene expression (SAGE; Sun et al., 2005 ).

Interestingly, a majority of differentially expressed genes in the left – right hemi- spheres function in regulating cell signaling or cell communication and in controlling gene or protein expression ( Sun, Collura, Ruvolo, & Walsh, 2006 ). The differential expression of candidate genes was further verifi ed in additional 12-week-old human fetal brains using either real-time reverse transcription polymerase chain reaction (RT- PCR) or in situ hybridization. Twenty-seven genes that show consistently higher expression either in the left or right hemisphere were identifi ed ( Sun & Walsh, 2006 ).

These studies provide two interesting fi ndings: (1) The human left and right hemi- spheres already have differential gene expression levels at early fetal stages (as early as 12 weeks), and (2) these differentially expressed genes may play a role in neurogen- esis and neural connections by regulating cell signaling and gene expression during early brain development. Identifying more differentially expressed genes in human left – right hemispheres and verifying their expression levels in human brains at differ- ent developmental stages will create a comprehensive picture of the molecular signa- ture of human brain asymmetry.

Additionally, genes critical in visceral organ asymmetries, such as molecules involved in SHH or NODAL pathways, are not identifi ed as differentially expressed in human fetal brains using this SAGE screening approach ( Sun et al., 2005 ). Previous work has shown that Shh and Nodal play a patterning role in visceral organ asym- metries at very early embryonic stages ( Capdevila et al., 2000 ; Hamada et al., 2002 ).

The earliest stage of the SAGE analyses is in human fetal 12-week-old brains. Differ- ential gene transcription between left – right hemispheres may have an early onset,

earlier than 12 weeks in the human fetus. Thus, even though molecules regulating body asymmetries might also be differentially expressed in human embryonic brains, they may not be detectable in 12-week-old human fetal brains. Whether brain asymmetry and body asymmetry share conserved signaling pathways still remains a question.

Differential gene transcription in a specifi c human brain region has also been detected recently. Using genome-wide screening approaches, such as cDNA microar- rays, 44 genes were identifi ed with enriched expression in frontal cortex and superior temporal regions of human fetal brains ( Abrahams et al., 2007 ). Gene coexpression relationships in specifi c human brain regions and cell types have also been carefully examined using microarrays ( Oldham et al., 2008 ). Even though these microarray analyses do not reveal genes that directly regulate brain asymmetry, gene transcription profi les generated in these studies will help us better understand human brain devel- opment and will provide databases for comparing brain development and evolution in humans and nonhuman primates ( Vallender, Mekel-Bobrov, & Lahn, 2008 ). More- over, the accumulation of gene expression profi les in different human brain regions from two hemispheres will allow comparisons of differential gene transcription in mirrored brain regions between left and right hemispheres.

Potential Mechanisms Regulating Brain Asymmetry

The screening approach has identifi ed many genes that are asymmetrically expressed in human fetal left and right hemispheres. Most of them are expressed in postmitotic cells based on their cellular functions ( Sun et al., 2006 ). Are there early initiating signals that are active in neural stem cells or progenitors between the embryonic left and right hemispheres? The initiating signals may be important in breaking early symmetry and in inducing asymmetrical expression of downstream genes that main- tain brain asymmetry ( Palmer, 2004 ).

The critical step of early development of the CNS is to form a tube structure called the neural tube ( Tanabe & Jessell, 1996 ). The forebrain (cerebral cortex) is located in the most anterior region of the neural tube. The potential molecular candidates that break the neural symmetry might be signals from the midline structures, such as the notochord and the fl oor plate ( Dodd, Jessell, & Placzek, 1998 ). In the forebrain, a structure anterior to the notochord is called the prechordal plate ( Rubenstein, Shimamura, Martinez, & Puelles, 1998 ). Secreting molecules such as Shh from the notochord or the prechordal plate may have uneven distribution between the left and right forebrain and break neural symmetry ( Tannahill, Harris, & Keynes, 2005 ).

Additionally, the roof plate in the neural tube is a resource for morphogens, for example BMPs and Wnts ( Chizhikov & Millen, 2005 ). The anterior neural ridge located in the most anterior cortical region transiently expresses the growth factor Fgf8 ( Fukuchi-Shimogori & Grove, 2001 ). Together, these secreted molecules, such as

BMPs, Wnts, and Fgf8, may play an early patterning role in the left and right forebrain asymmetry.

Recent studies have demonstrated that different cell division rates are observed in distinct regions of the primate and mouse cortices and may contribute to the forma- tion of different cortical functional areas ( Lukaszewicz et al., 2005 ; Polleux, Dehay, Moraillon, & Kennedy, 1997 ). Is cortical asymmetry simply a result of different pro- liferation rates of progenitors and, subsequently, temporal and spatial difference of neural differentiation and connections between the two hemispheres? One important function of morphogens, such as Shh, BMPs, and growth factors, is to regulate prolif- eration. The uneven distribution of morphogens between two hemispheres may cause different proliferation rates. Differentially expressed genes in human fetal 12- to 19- week-old brains, identifi ed by the SAGE screening approach ( Sun et al., 2005 ), may refl ect the temporally and spatially distinct neuronal differentiation and connections between the two hemispheres. Because consistent asymmetry is mostly detected in human brains, testing these potential mechanisms of brain asymmetry directly in humans remains a challenge.

Detecting Differential Gene Expression in Nonhuman Brains

While it is diffi cult to directly test the molecular regulation of brain asymmetry using experimental approaches in humans, revealing differential gene expression and mech- anisms of laterality in nonhuman brains is favorable.

Biased hand use is also observed in nonhuman primates, such as chimpanzees.

Because most studies on preferential hand use have entailed investigating manual tasks of captive great apes, whether there is consistent handedness at a popula- tion level in wild apes is arguable ( Hopkins & Cantalupo, 2005 ; McGrew, Marchant, Wrangham, & Klein, 1999 ). A recent study of wild chimpanzees living in the Gombe National Park has provided some new insights into hand preferences in nonhuman primates. It appears that handedness in wild chimpanzees varies depending on the type of tool use and the specifi c manual task ( Lonsdorf & Hopkins, 2005 ). These variations make it diffi cult to identify genes that may be associated with handedness in nonhuman primates.

Interestingly, several recent studies have shown asymmetries of the planum tem- porale and Broca ’ s area in the brains of chimpanzees and great apes ( Cantalupo &

Hopkins, 2001 ; Gannon, Holloway, Broadfi eld, & Braun, 1998 ; Hopkins, Marino, Rilling, & MacGregor, 1998 ). These anatomical asymmetries in the simian brains resemble those of humans. Even though the functional relevance of simian brain asymmetry, for example, related to “ language ability, ” remains a question ( Hutsler &

Galuske, 2003 ), genes that contribute to the asymmetrical structural formation in the primate cerebral cortex may have similar roles in regulating asymmetries in human

brains. Thus, using primate brain tissues may help us identify differentially expressed genes, map their expression patterns, and gain insights into conserved gene expression and function in human brain tissues. However, the diffi culty of developing genetic primate models hurdles further examination of gene functions in brain asymmetry using primates.

The rodent is an ideal animal model for studying brain development and functions because of low cost, well-developed genetic tools, and established behavioral tests.

However, consistent anatomical and functional brain asymmetries have not been well described in rodents. A biased food reaching task, called paw preference, is often observed in an individual mouse with preferential use of either the left or right front paw. However, paw preference becomes randomly distributed at a population level ( Biddle, Coffaro, Ziehr, & Eales, 1993 ; Signore et al., 1991 ). By breeding mice that exhibit consistent paw preference, mouse lines showing strong lateralized food reaching have been created ( Collins, 1991 ). However, these mice still do not have a consistent 9:1 ratio of preferential hand use at a population level as reported in humans. These studies indicate that a genetic program of brain asymmetry may exist in rodent brains but it is not preferentially biased to either the left or right hemisphere. Nevertheless, since the techniques of creating transgenic and gene mutant mice are becoming conventional in many laboratories, examining functions of differentially expressed genes, detected in human and primate brains, in mouse models is a promising direction for understanding the genetic mechanisms of brain asymmetry.

The epithalamus, a structure of the diencephalon, consists of the habenula and the pineal complex and shows left-side asymmetries in zebra fi sh. The molecular regula- tion of the epithalamus asymmetry in zebra fi sh has been revealed ( Concha & Wilson, 2001 ; Halpern, Liang, & Gamse, 2003 ). Consistent with the role of the Nodal pathway in the molecular regulation of body asymmetry, Nodal-related genes Cyclops and Nodal downstream genes lefty1 and pitx2 have been shown to control the asymmetry of the diencephalon in the zebra-fi sh brain ( Bisgrove, Essner, & Yost, 2000 ; Concha, Burdine, Russell, Schier, & Wilson, 2000 ; Essner, Branford, Zhang, & Yost, 2000 ; Halpern et al., 2003 ). Developing behavioral tests relevant to the epithalamus asym- metry in zebra fi sh will help us better understand the signifi cance of biological functions of asymmetrical gene expression in zebra-fi sh brains.

Moreover, lateralized song production has been observed in songbirds. Denervation of the left syrinx, the vocal organ of songbirds, has been shown to result in the loss of 90% – 95% of song syllables in canaries ( Hartley & Suthers, 1990 ; Nottebohm, Manning, & Nottebohm, 1979 ; Nottebohm & Nottebohm, 1976 ). Lesions to the high vocal center on the left canary cortex, but not the right, also cause dramatic loss of song components ( Nottebohm, Stokes, & Leonard, 1976 ). It appears that canaries display lateralized song production, with dominance on the left side. It is unclear

whether genetic programs contribute to this laterality in songbirds. Since brain tissues for songbirds are relatively easy to collect at different developmental stages, identify- ing genes that are differentially expressed in left and right hemispheres may provide insights into lateralized song production. Moreover, since the neurocircuitry of song production in songbirds is well understood ( Bottjer, Halsema, Brown, & Miesner, 1989 ; Nottebohm, Kelley, & Paton, 1982 ; Nottebohm et al., 1976 ), developing techniques to alter gene expression in songbird brains can further reveal how the lateralized song system is established.

Approaches to Identifying Genes Involved in Human Brain Asymmetry Forward Genetic Approach

A forward genetic screen is based on the identifi cation of mutated genes that produce certain phenotypes. Mapping genes that are associated with disrupted body and brain asymmetries in humans may help us identify the genetic controls that lead to normal asymmetries. Complete reversal of normal organ position, such as heart and lungs, is called situs inversus (SI). Anatomic and functional MRI studies have shown that the left-hemisphere dominance for language is detected in individuals with SI as frequently as in normal subjects ( Kennedy et al., 1999 ). Moreover, Kartagener ’ s syndrome, a disorder caused by cilia with decreased or total absence of motility, represents 50% of SI cases ( Carl é n & Stenram, 2005 ). However, these SI individuals with Kartagener ’ s syndrome are found with normal ratio of dominance for right hand- edness ( McManus, Martin, Stubbings, Chung, & Mitchison, 2004 ). These studies suggest that individuals with abnormal body asymmetry can have normal asymmetri- cal brain function and handedness. It appears that the genetic regulation of visceral organ asymmetries may be distinct from that of brain asymmetries and handedness ( Hobert, Johnston, & Chang, 2002 ; C. McManus, 2005 ). Therefore, identifying genes involved in visceral organ asymmetries using forward genetic approaches may not help us understand the molecular mechanisms of human brain asymmetry.

Directly mapping genes that are associated with disrupted brain asymmetry may be a better approach to identifying the molecular regulation of brain laterality in humans. Reduced and reversed anatomical brain asymmetry has been reported in individuals with schizophrenia, autism, and dyslexia ( Falkai et al., 1992 ; Galaburda, Menard, & Rosen, 1994 ; Herbert et al., 2005 ; Hugdahl et al., 1998 ). Whether abnormal brain asymmetry is the cause or result of these neurological disorders is unknown.

Recently, research into the causal relationships between genes and autism has made promising progress ( Geschwind, 2008 ; Walsh, Morrow, & Rubenstein, 2008 ). Examin- ing functions of candidate genes of autism may provide insight into not only the causes of autism but also functional connections of these genes to brain asymmetrical development.

Additionally, several recent case reports have shown that polymicrogyria (PMG), characterized by an excessive number of small gyri in the brain, occurs only on one side of the cortex, called unilateral polymicrogyria ( Chang et al., 2006 ; Pascual- Castroviejo, Pascual-Pascual, Viano, Martinez, & Palencia, 2001 ). The presence of several pedigrees in which the disorder is present in more than one individual of affected families suggests that unilateral PMG appears to be caused by gene mutations ( Chang et al., 2006 ). Mapping genes involved in unilateral PMG using forward genetic approaches may reveal their role in normal formation of brain asymmetry in humans.

Microarrays

Microarray is a method to generate high throughput gene expression profi les. To detect gene expression levels, DNA oligonucleotides corresponding to various regions of a specifi c gene are attached to glass slides. Slides are hybridized with fl uorescence- labeled complementary DNAs (cDNAs) or complementary RNAs (cRNAs) and scanned with fl uorescent readers. Usually the slides are hybridized simultaneously with cDNAs/

cRNAs derived from two samples, for instance, with samples from the left and right hemispheres. The fl uorescence intensities for each cDNA are quantifi ed and compared between two samples. Gene transcripts that are either up- or down-regulated in the left or right hemisphere can be detected. Thus, microarray is an effective method for measuring expression levels of known genes between samples. Microarrays have been used to comprehensively characterize distinct gene expression in different brain regions in both mice and humans ( Oldham et al., 2008 ; Sunkin & Hohmann, 2007 ).

Using the microarray technique, one can identify genes with differential transcriptions between left and right hemispheres of human brains.

Serial Analysis of Gene Expression

SAGE is a method for the comprehensive analysis of gene expression levels without the prior knowledge of gene transcripts. SAGE has been successfully used to identify genes with differential expression levels in specifi c tissues in many systems, including human fetal left and right brains ( Blackshaw, Fraioli, Furukawa, & Cepko, 2001 ; Sun et al., 2005 ; Velculescu, Zhang, Vogelstein, & Kinzler, 1995 ; Ye, Usher, & Zhang, 2002 ).

The advantages of using SAGE versus microarrays are as follows: (1) SAGE can detect transcripts at low expression levels that may be undetectable by microarrays, and (2) SAGE can easily compare gene expression levels among multiple samples. For instance, once SAGE libraries are created from the left and right hemispheres of developing human cortices, they can be compared at different developmental stages in one side of the brain or between left and right hemispheres. To reveal the evolutionary mech- anisms of brain asymmetry, SAGE has been used for the comparison of gene expression levels across species — for example, between human and primate brains ( Sun et al., 2006 ).