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Early Asymmetry of Gene Transcription in Embryonic Human Left and Right Cerebral Cortex

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Science  17 Jun 2005:
Vol. 308, Issue 5729, pp. 1794-1798
DOI: 10.1126/science.1110324

Abstract

The human left and right cerebral hemispheres are anatomically and functionally asymmetric. To test whether human cortical asymmetry has a molecular basis, we studied gene expression levels between the left and right embryonic hemispheres using serial analysis of gene expression (SAGE). We identified and verified 27 differentially expressed genes, which suggests that human cortical asymmetry is accompanied by early, marked transcriptional asymmetries. LMO4 is consistently more highly expressed in the right perisylvian human cerebral cortex than in the left and is essential for cortical development in mice, suggesting that human left-right specialization reflects asymmetric cortical development at early stages.

One of the most remarkable aspects of the human cerebral cortex is that the two hemispheres are specialized for distinct cognitive and behavioral functions. Whereas the right cerebral cortex regulates movement of the left side of the body and vice versa, ∼90% of the human population is naturally more skilled with the right hand than with the left (1). This motor asymmetry is strongly correlated with language dominance: Language function is predominantly localized to a distributed network in the left perisylvian cortex in 97% of right-handers and ∼60% of left-handers (2, 3). Functional asymmetries exist in mathematical ability and in spatial and facial recognition as well. These functional asymmetries have been related to anatomical asymmetries of the cortex that are somewhat more subtle (2, 4). For example, the posterior end of the sylvian fissure is higher in the right hemisphere than in the left (5). The planum temporale, a region in the posterior portion of the superior temporal sulcus in which Wernike's area resides, is larger in the left hemisphere than in the right in more than 65% of examined adult and 56 to 79% of examined fetus and infant brains, so the anatomical asymmetries are less marked than the functional ones (6, 7). Although genetic factors connecting cerebral asymmetry and functional dominance have been supported (8), no molecular correlate of cerebral asymmetry has been identified.

We directly tested the hypothesis that left-right cortical asymmetry in humans results from differential gene expression at early embryonic stages, long before the onset of organized cerebral cortical function. By applying serial analysis of gene expression (SAGE), we measured gene expression levels between the left and right hemispheres in early (12- to 14-week-old) fetal human brains, during periods of neuronal proliferation and migration, and later (at 19 weeks), after these processes are largely completed (9). Brain tissues were first dissected from matching perisylvian regions in two hemispheres (Fig. 1, A to C). The cortex was then separated at the midline. On the medial side of the hemisphere, tissues were also dissected from the ventricular zone in the frontal and occipital regions (Fig. 1B). Total RNA was isolated, and 14 SAGE libraries were generated (Fig. 1D). To detect genes with differential expression levels, we compared the tag frequency for each gene between two SAGE libraries generated from the frontal, perisylvian, and occipital regions in the left-right hemispheres. To verify the statistical significance of differences in each comparison, we performed a Monte Carlo test and verified this using the χ test. Using the χ-squared distribution with one degree of freedom and confidence levels (P), we sorted genes within each comparison (e.g., left-right). A higher χ value indicates a greater statistically significant difference.

Fig. 1.

Dissection of human embryonic brain tissues and generation of human SAGE libraries. (A) A top view of a 14-week-old human embryonic brain. Tissues were dissected from perisylvian regions in the left (L) and right (R) hemispheres. (B) A side view of a 14-week-old human embryonic right hemisphere. Tissues in the frontal (f, red) and occipital (o, blue) ventricular regions containing dividing cells were dissected. The dorsal cortex (d) is on the top. (C) The left side view of a 14-week-old human embryonic brain. The perisylvian region is circled. (D) Summary of human brain SAGE libraries. The male (M) and female (F) brains are listed. At least 55,000 tags were sequenced in each library. (E) Summary of differentially expressed genes detected by SAGE analysis between the left-right hemispheres. Numbers of genes that are highly expressed in the left (L > R) or right (L < R) hemisphere are listed with confidence levels P > 99% and 95% < P < 99%. n/d, not detected.

In all, 49 differentially expressed genes were identified by SAGE with P > 99% (χ value > 6.63) between the left-right perisylvian regions of a 12-week-old embryonic human cortex. Among them, 21 genes were highly expressed in the left region, whereas 28 genes were highly expressed in the right (Fig. 1E). Moreover, 68 genes were identified with P > 99% between the left-right perisylvian regions of a 14-week-old cortex (Fig. 1E). By combining analyses, we generated a list of statistically differentially expressed genes between the left-right hemispheres in the perisylvian regions (Fig. 1E and tables S1 to S5) and frontal and occipital regions (tables S9 to S12) of human embryonic brains at 12, 14, and 19 weeks. Differential gene expression levels detected by SAGE suggested an early transcriptional asymmetry between the left-right hemispheres in human embryonic brains.

One of the genes reproducibly asymmetrically expressed was the transcription factor Lim Domain Only 4 (LMO4). Using SAGE analysis, we found that the human LMO4 is more highly expressed in the perisylvian regions of the right hemisphere than in the left at both 12 and 14 weeks (Fig. 2A). In contrast, LMO4 expression levels did not show significant differences between the left and right perisylvian regions at 19 weeks (Fig. 2A). We then quantified LMO4 expression levels using real-time SYBR (Applied Biosystems)–green reverse transcription–polymerase chain reaction (RT-PCR) and confirmed higher LMO4 expression in the right perisylvian regions than in the left of embryonic 12- and 14-week-old but not 19-week-old brains, using the same RNA samples for SAGE analysis (Fig. 2B). Moreover, we confirmed higher levels of LMO4 expression in the right perisylvian region versus the left in second 12-week-old and 14-week-old brains and observed modest differences in two 16-week-old brains and one 17-week-old brain (Fig. 2B).

Fig. 2.

Human LMO4 was highly expressed in the right hemisphere as detected by SAGE, real-time RT-PCR, and in situ hybridization. (A) The human LMO4 expression levels in the perisylvian regions measured by SAGE (tag frequencies) in 12-, 14-, and 19-week-old brains. wk, week. (B) The LMO4 expression levels between the left and right hemispheres were verified by real-time RT-PCR in eight human embryonic brains (at 12 to 19 weeks). Two data points from duplicated experiments for each sample are illustrated. (C to F) LMO4 expression in coronal sections cut from the frontal (f) to occipital (o) lobes of a human embryonic 12-week-old brain. The medial-lateral extent of LMO4 expression in the cortical plate between the left (white arrowheads) and right (black arrowheads) hemispheres was defined by a red line connecting the corticostriatal sulcus (cs) and the lateral border of the putamen (arrow). Numbers in (D) through (F) indicate the sections shown in (C), (G to L). Human LMO4 was more highly expressed in the cortical plates in the right hemispheres than in the left in coronal sections of [(G) to (J)] a 14-week-old brain and [(K) and (L)] a 16-week-old brain. (H), (J), and (L) show high-power views of selected areas in (G), (I), and (K), respectively. (G) and (I) illustrate two different rostral-caudal levels through the frontal lobe and presumptive perisylvian region. The dorsal (d) and ventral (v) areas of the cortex are labeled.

We next performed nonradioactive in situ hybridization on human embryonic brains and noted right-left differences in the extent of LMO4 expression at 12 weeks. We serially sectioned the cortex in the frontal plane and performed in situ hybridization on at least 54 sections from this series, covering most of the frontal to occipital extent of the cerebral cortex (Fig. 2C). Consistent with the early expression of Lmo4 in mice (see below), LMO4 in this 12-week-old human brain was expressed in the ventral lateral cortical plate in a patchy fashion (Fig. 2, D to F). LMO4 was also expressed highly and symmetrically in noncortical telencephalic structures, notably the putamen (Fig. 2F). We analyzed the medial-lateral extent of LMO4 expression in the cortical plate in relation to the lateral border of the basal ganglia, defined by connecting the corticostriatal sulcus and the lateral border of the putamen (Fig. 2F). In this brain, LMO4 expression was observed further dorsolateral in the cortical plate in the right hemisphere than the left, particularly in sections near the future perisylvian region (Fig. 2F).

We then confirmed asymmetric LMO4 expression in several additional human fetal brains, focusing in the perisylvian region, using [35S]-labeled radioactive in situ hybridization. At 14 weeks, LMO4 was highly expressed in the cortical plate around the entire perimeter of the cortex. Although levels of LMO4 expression in the right hemisphere were comparable to those in the left in broad areas of dorsomedial neocortex, paralleling the fact that there are no known anatomical asymmetries of these medial areas, it was consistently more highly expressed over broad areas of the right perisylvian cortex than the left (Fig. 2, G to J). At 16 weeks, asymmetric cortical expression was still observed, although it was diminished relative to earlier time points, consistent with the real-time RT-PCR analysis (Fig. 2, B, K, and L). Marked asymmetries in LMO4 expression were seen in the perisylvian region of a human 17-week-old cortex studied by nonradioactive in situ hybridization (fig. S1) and were even more apparent than in the RT-PCR results (Fig. 2B). In a 19-week-old brain, consistent with RT-PCR results, left-right differences in LMO4 expression were not obvious (fig. S1). Overall, the in situ hybridization analysis mirrored the SAGE and RT-PCR analysis, with clearer but variable asymmetries in expression among individuals at earlier stages and no clear asymmetry at the latest stage examined (19 weeks).

To better understand the dynamic change in Lmo4 expression during cortical development, we analyzed Lmo4 expression in mouse brains. Similar to its expression in the 12-week-old human embryonic brain, Lmo4 was weakly expressed in the ventral cortex in the embryonic day 11.5 (E11.5) mouse brain, and its expression increased during development (Fig. 3A). Lmo4 expression boundaries were fairly sharp at postnatal day 1 (P1) with expression being high in the anterior and posterior portions of the cortex, but with a large zone of nonexpression that overlapped the presumptive parietal cortex in between (Fig. 3D). However, this nonexpression zone disappeared at P17 (Fig. 3G). In coronal sections of E15.5 and P5 mouse cortices, Lmo4 was expressed in the medial and lateral cortical areas as well (Fig. 3, B and E). Lmo4 in the mouse showed apparent asymmetries in the cortical area in which it was highly expressed, and the expression pattern was quite dynamic (Fig. 3, A to G).

Fig. 3.

The dynamic and asymmetric expression of Lmo4 in mouse brains. (A to F) The patchy and asymmetric expression patterns of Lmo4 are illustrated in [(A) to (C)] coronal sections of mouse brains from (A) E11.5 and (B) E15.5; (D) whole mount in situ hybridization from P1; and (E and F) coronal sections of P5 brain. (C) and (F) show high-power views of Lmo4 expression in the cortical plates (cp) (red stars) in selected areas from (B) and (E), respectively. The dorsal cortex is on the left of (C) and (F). (G) Lmo4 expression appears uniform in the cortical plate in a sagittal section of a P17 brain. (H) Schematic view of the forebrain from above, indicating the sagittal section shown in (I). (I) Lmo4 expression was divided into the anterior (a) (Expression I), the posterior (p) (Expression II), and the nonexpression regions in sagittal sections of P1 mouse cortex. The dorsal (d) and ventral (v) areas of the cortex are labeled. (J and K) Asymmetric expression of Lmo4 in the left-right hemispheres in representative P1 mouse cortices. The ratio of the size of each domain in the left versus the right hemisphere is plotted for serial sagittal sections. In total, six brains were similar to that in (J) and four brains to that in (K).

Because the levels of coronal sections may affect in situ hybridization signal, we mapped the Lmo4 expression on serial sagittal sections to provide an accurate gene expression pattern (Fig. 3H). We divided the cortical expression of Lmo4 in P1 brain into three regions: anterior (expression I), intermediate (nonexpression), and posterior (expression II) (Fig. 3I). The ratios of the length of each Lmo4 expression region versus the full length of the cortex were calculated from the medial to lateral cortical regions and analyzed with histograms (Fig. 3, J and K). The Lmo4 expression areas in the anterior were smaller in the left hemisphere than in the right in six tested brains (shown by one representative brain in Fig. 3J), but larger in the left than the right in four tested brains (shown by one representative brain in Fig. 3K). Correspondingly, the Lmo4 nonexpression areas (intermediate) were larger in the left hemisphere than in the right in the same six tested brains (Fig. 3J) but smaller in the same four tested brains (Fig. 3K). However, we did not detect asymmetric Lmo4 expression in the posterior cortex between the left-right hemispheres in any of the tested brains (Fig. 3, J and K). Thus, although Lmo4 expression in mouse cortex was moderately asymmetrical in every individual brain tested so far, it was not consistently lateralized to the right or left side. This may relate to behavioral and anatomical studies in mice, in which sensory-motor asymmetries, like paw preference, are observed in individual mice but are not biased on a population level to either the right or left hemisphere, as hand preference is in humans (1014). The differences in mice and humans suggest the possibility that paw preference in rodents might reflect an early, perhaps stochastic, developmental asymmetry that is established perinatally, before paw usage, implying a transcriptional asymmetry that is not consistently lateralized to the left and right. Evolution of mechanisms that bias or entrain a modest and random asymmetry in lower organisms may have allowed the development of more consistent functional asymmetries in the human cortex (15).

To identify other differentially expressed genes between the left-right hemispheres, we focused on genes showing different expression levels measured by SAGE in the perisylvian regions of human embryonic 12-week-old brains. Using RNA samples for generating SAGE libraries, we verified 76 genes using real-time RT-PCR (tables S6 and S7) and found 39 genes (51%) showing consistent differential expression as measured by SAGE (table S6). To further test the reproducibility of verifying SAGE data, we verified expression levels of these 76 genes using a second 12-week-old brain. We found that 27 genes (36%) consistently showed differential expression (either left-high or left-low) in both brain samples (table S6). We also tested 17 genes that have lower χ values (<1.9) but have been implicated in cortical development and found 7 genes showing relatively significant differences of gene expression levels, although they were not detected by SAGE (table S8).

The left-right differences in LMO4 expression in humans could potentially reflect either a differing topographic mapping in the two hemispheres or a difference in the tempo of cortical development, with the right hemisphere's development leading over the left. In mice, Lmo4 expression marks anterior and posterior regions in the mouse cortex and is shifted in cortices of Pax6 and Fgf8 mutants, consistent with the suggestion that Lmo4 expression at P0 to P5 reflects overall cortical topographic mapping (fig. S2). On the other hand, there is some evidence for the appearance of the several cortical sulci and gyri at earlier ages in human right hemisphere than in the left, for instance, the rolandic sulcus (which appears at 17 to 20 weeks) and the superior temporal fissure (at 23 weeks) (2, 7, 16). Thus, higher LMO4 expression in the right than the left hemisphere could reflect the arrival of corresponding developmental stages sooner in the right than the left hemisphere. Either model, however, implies molecular events that greatly precede morphological asymmetries and provide potential insight into the mechanism of generation of asymmetry. Furthermore, the molecular events that regulate LMO4 expression in humans may be secreted molecules, such as FGF8, and/or gradients in transcription factors in the ventricular zone, such as PAX6 and EMX2, as in mice. Indeed, some factors with potential roles in cortical development, such as ID2 and NEUROD6, were asymmetric in SAGE and/or RT-PCR analyses in human embryonic 12-week-old brains (tables S6 and S8). Understanding the factors that regulate LMO4 expression may ultimately identify earlier events in left-right brain asymmetry. On the other hand, our results generally confirm the earlier suggestion that genes previously implicated in visceral asymmetries are not detectably implicated in cerebral asymmetries (3, 17). For instance, mutations that result in “situs inversus” in humans do not appear to disrupt the left-hemisphere localization of language, mathematical, and hearing abilities and handedness (18). Except possibly for the FGF signaling pathway, we did not detect significant differences of expression levels of genes regulating body asymmetry in the human embryonic SAGE libraries, although asymmetries at earlier developmental stages cannot be ruled out (3, 19).

Abnormalities of cerebral cortical asymmetry have been reported in a wide array of neuropsychiatric disorders, such as autism, schizophrenia, and dyslexia (2023). The presence of early asymmetries in gene transcription in the two cerebral hemispheres thus provides potential pathways through which a number of developmental disorders may ultimately converge on the abnormal development of human cerebral asymmetry.

Supporting Online Material

www.sciencemag.org/cgi/content/full/1110324/DC1

Materials and Methods

Figs. S1 and S2

Tables S1 to S26

References and Notes

References and Notes

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