Postmitotic regulation of sensory area patterning in the mammalian neocortex by Lhx2 Andreas Zembrzyckia, Carlos G. Perez-Garciaa, Chia-Fang Wangb, Shen-Ju Choub, and Dennis D. M. O’Learya,1 a Molecular Neurobiology Laboratory, The Salk Institute for Biological Studies, La Jolla, CA 92037; and bInstitute of Cellular and Organismic Biology, Academia Sinica, Taipei, 115, Taiwan

Current knowledge suggests that cortical sensory area identity is controlled by transcription factors (TFs) that specify area features in progenitor cells and subsequently their progeny in a one-step process. However, how neurons acquire and maintain these features is unclear. We have used conditional inactivation restricted to postmitotic cortical neurons in mice to investigate the role of the TF LIM homeobox 2 (Lhx2) in this process and report that in conditional mutant cortices area patterning is normal in progenitors but strongly affected in cortical plate (CP) neurons. We show that Lhx2 controls neocortical area patterning by regulating downstream genetic and epigenetic regulators that drive the acquisition of molecular properties in CP neurons. Our results question a strict hierarchy in which progenitors dominate area identity, suggesting a novel and more comprehensive two-step model of area patterning: In progenitors, patterning TFs prespecify sensory area blueprints. Sequentially, sustained function of alignment TFs, including Lhx2, is essential to maintain and to translate the blueprints into functional sensory area properties in cortical neurons postmitotically. Our results reemphasize critical roles for Lhx2 that acts as one of the terminal selector genes in controlling principal properties of neurons. terminal selector genes CoupTF1

| epigenetic mechanisms | neuronal fate | MeCP2 |

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he adult mammalian cortex is patterned into distinct and modality-specific sensory areas that are responsible for the perception of the sensory information and for the control of behavior (1). Research has focused in particular on how transcription factors (TFs), which are expressed in gradients in the cortical ventricular zone (VZ) progenitors, drive area patterning of mature cortical sensory areas by specifying their size and position during early cortical development (1, 2). As a result, current views suggest that the specification of sensory area identity is dominated by patterning events in progenitors (1–3). However, the mechanisms that translate area-patterning information from cortical progenitors into area-specific properties of postmitotic (CP) neurons are not well understood. Results We hypothesized that sensory area identity in CP neurons in mice could be determined ultimately by the function of key regulators that function postmitotically. One of the few TFs that are expressed in progenitors and neurons is LIM-homeodomain 2 (Lhx2) (4). During early corticogenesis, Lhx2 shows a caudal/ medial-high to rostral/lateral-low expression gradient in cortical progenitors. Starting from around embryonic day (E) 12 to postnatal day (P) 0, a caudal/lateral-high to rostral/medial-low expression gradient is apparent in cortical neurons (Fig. S1), which postnatally becomes restricted to more uniform expression in upper cortical layers (Fig. S1). Such sustained and dynamic expression of Lhx2 in neurons suggests that Lhx2, similarly to its established roles exerted in progenitors (4–8), may affect properties in cortical neurons. To test this hypothesis, we used conditional inactivation by crossing mice carrying floxed Lhx2 alleles (4) with mice expressing Nex-Cre mediating Cre recombination restricted to postmitotic cortical neurons (9) starting around E11 (Fig. S2). www.pnas.org/cgi/doi/10.1073/pnas.1424440112

Immunostaining using an Lhx2 antibody at E13 and E17 reveals that in conditional knockout Lhx2flox/flox-NexCre+, (hereafter referred to as “cKO”) specimens Lhx2 expression remains unchanged in the VZ and subventricular zone (SVZ) but is absent in the intermediate zone and CP, demonstrating selective deletion of Lhx2 in cortical postmitotic neurons (Fig. S2). At P7, we compared dissected WT and cKO brains and analyzed their neuroanatomy using Nissl staining and found no differences, showing that overall brain development, cortical anatomy, and thickness of cortical layers is indistinguishable among genotypes (Fig. S2). Contrary to deletion of Lhx2 in progenitors (4–8), our data demonstrate that gross cortical development is not affected by postmitotic deletion of Lhx2. This result allowed us to investigate processes that mature postnatally such as area patterning. Lhx2 Controls Thalamocortical Connectivity. We identified primary sensory area borders by serotonin (5HT) immunostaining that in rats and mice during early postnatal development labels the cortical terminations of subsets of thalamocortical axons (TCAs) of relay neurons that project from the principal thalamic sensory nuclei to layer 4 of the corresponding primary cortical sensory area (10–12). Subsequently, we quantified sensory area dimensions on flattened horizontal P7 cortex sections (Fig. 1A). Measured mean values for cKO specimens are displayed as percent of WT, and the variance (±) among the tested specimens was calculated using SEM. Despite comparable cortical surface area (97 ± 0.93%, P = 0.0529) and frontal/motor cortex (M) area (104.4 ± 2.4%, P = 0.1002), 5HT staining showed that size and shape of sensory areas is affected in an opposing fashion in cKO sections.

Significance The mammalian neocortex is divided into specialized modalityspecific areas that are responsible for the processing of sensory information. This architecture is critical, because altered area size affects normal sensory function and behavior in animals and humans. Current knowledge suggests that sensory area specification is dominated by patterning genes expressed in cortical progenitors. We show that postmitotic deletion of the transcription factor LIM homeobox 2 (Lhx2) in cortical neurons does not affect area patterning in progenitors but strongly alters sensory areas, demonstrating that specification of area identity in progenitors alone is insufficient. We suggest a novel and more comprehensive model of cortical area patterning that incorporates these revelations and define the relevance of postmitotic mechanisms in determining the functional properties of cortical areas. Author contributions: A.Z. and D.D.M.O. designed research; A.Z., C.G.P.-G., C.-F.W., and S.-J.C. performed research; S.-J.C. contributed new reagents/analytic tools; A.Z., C.G.P.-G., S.-J.C., and D.D.M.O. analyzed data; and A.Z., C.G.P.-G., S.-J.C., and D.D.M.O. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. 1

To whom correspondence should be addressed. Email: [email protected].

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1424440112/-/DCSupplemental.

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Edited by Carla J. Shatz, Stanford University, Stanford, CA, and approved April 16, 2015 (received for review December 20, 2014)

Fig. 1. Postmitotic deletion of Lhx2 alters thalamic input and the cortical molecular profile. (A) 5HT immunostaining on P7 flattened cortex (CTX) sections reveals the terminations of TCAs that project from the principal sensory thalamic nuclei into cortical layer 4. In cKO (Lhx2flox/flox-NexCre+) brains, the 5HT staining reveals abnormal cortical sensory areas, compared with WT brains. Quantification of sensory area size (dotted lines, displayed as percent of WT) reveals that cortical surface (97 ± 0.93%, P = 0.0529) and the M area (104.37 ± 2.4%, P = 0.1002) are comparable in WT and cKO brains. V1 is enlarged (171.09 ± 2.39%, P < 0.0001) in cKO brains, whereas S1 (65.14 ± 1.81%, P < 0.0001) and A area (54.31 ± 4.69%, P < 0.0001) are contracted. *P < 0.05. (B) ISH for RORb, Cad8, and EphA7 was performed on P7 flattened cortex sections at depths comparable to 5HT-stained sections. The expression patterns of molecular markers appear superimposable to the borders of 5HT staining (compare outlines). n, number of samples; PMBSF, posterior medial barrel subfield in S1. Main axes: A, anterior; L, lateral; M, medial; P, posterior. (Scale bars: 0.5 mm.)

For example in cKO brains the visual cortex (V1) expanded massively (171.1 ± 2.4%, P < 0.0001), whereas auditory cortex (A) and somatosensory cortex (S1) were severely contracted (A: 54.3 ± 4.7%, P < 0.0001; S1: 65.1 ± 1.8%, P < 0.0001), revealing that targeting of TCAs from the principal thalamic nuclei into cortical layer 4 (L4) is strongly affected. To investigate the altered sensory TCA connectivity in more detail, we performed lipophilic dye-tracing experiments at P7 using an unbiased approach. Different dyes were inserted in defined locations on the cortical sheet that across brains correspond to stereotaxically very similar locations (Fig. S3). First, we inserted DiI (1,1′-dioctadecyl 3,3,3′,3′-tetramethylindocarbocyanine perchlorate; red dye) and DiO (3,3′-dioctadecyloxacarbocyanine perchlorate; green dye) crystals into a cortical location that presumably shows retained 5HT staining (Fig. 1A: approximated center regions of S1 and V1). The retrogradely DiI-labeled cell bodies (red) were indeed evident only in the somatosensory thalamic nucleus (ventroposterior nucleus: VPN), whereas retrogradely DiO-labeled cell bodies (green) were found exclusively in the visual thalamic nucleus (dorsal lateral geniculate nucleus, dLG) in WT and cKO coronal sections of the thalamus (Fig. S3A). To reveal the rostral expansion of V1 at the expense of S1 territory demonstrated by 5HT staining in cKO cortices (Fig. 1A), we 2 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.1424440112

performed an additional dye-labeling approach and placed DiI (red dye) into approximated cortical locations that in WT correspond to the rostral V1 region and placed DiD (1,1’-dioctadecyl3,3,3′,3′-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt; green dye) into the presumptive caudal S1 region (Fig. S3B). Across genotypes, DiI retrogradely labeled cells (red) in the dLG. In WT sections, DiD retrogradely labeled TCAs (green) that avoid the dLG and exclusively labeled cells in the VPN. Conversely in cKO sections, DiD-labeled TCAs ectopically labeled cells in the dLG. Only some cells in the VPN were labeled with DiD indicating that most of the cortical territory around the DiD injection site had obtained V1 instead of S1 identity. These results are consistent with abnormal 5HT staining (Fig. 1A) showing that subcortical TCA input from the principal sensory thalamic nuclei into cortical layer 4 is altered in cKO brains. In the cortex, complementary and area-specific gene expression distinguishes areas from one another molecularly (1–3). To analyze if altered TCA targeting in cKO coincides with altered L4 molecular markers, we performed 5HT staining and in situ hybridization (ISH) on P7 flattened horizontal cortical sections that cover L4 (Fig. 1B) and show 5HT staining (10–12). We report that high RORb (RAR-related orphan receptor B) expression domains delineate V1, S1, and A, mirroring the 5HT staining pattern (compare 5HT and RORb in Fig. 1B). High Cad8 (Cadherin 8) expression in the rostral cortex delineates the M/S1 border, whereas low expression delineates V1 and S1. Low EphA7 (Eph receptor A7) expression domains similarly delineate V1 and auditory areas. In cKO, the overall expression levels of these molecular markers appear normal, but their expression boundaries are abnormal, mimicking the altered 5HT patterns (Fig. 1B) and thus showing that Lhx2 regulates the area-specific molecular profile and the targeting of TCAs in cortical neurons that correspond to layer 4. Lhx2 Controls Area Patterning Across Layers in the CP. Next, we analyzed the developmental dynamics of Lhx2 expression in cortical neurons. We performed double immunostainings using an Lhx2 antibody together with antibodies for layer-specific neuronal markers between E12 and P7. In layers 2–4 at P7, Lhx2 staining overlaps with the upper cortical layer marker Satb2 (special ATrich sequence binding protein 2) (Fig. S4A). This overlap is consistent with coexpression of Lhx2 in Tbr2-positive (Eomes, T-box brain protein 2) basal progenitors at E15 (Fig. S4B), which generate these neurons (13). Conversely at P7, Lhx2 shows no overlapping staining with Ctip2-positive layer 5 neurons (chicken ovalbumin upstream promoter transcription factor-interacting protein 2) (Fig. S4A). However, Lhx2 is transiently coexpressed in most newly born Ctip2-positive layer 5 neurons at E14–15 (Fig. S5) as well as in most newly born Tbr1-positive (T-box brain protein 1) layer 6 neurons at E12–13 (Fig. S6). This result demonstrates that CP neurons express Lhx2, either transiently after they are born (lower-layer neurons) or permanently (upper-layer neurons; also see the schematic in Fig. S7), and suggests that Lhx2 function could control molecular properties broadly across all cortical layers. Subsequently, we investigated whether area-specific geneexpression borders in cKO cortices are broadly affected across layers in the CP by analyzing the expression of area-specific and layer-specific marker genes using ISH on P7 sagittal sections (Fig. 2A). As on stained tangential cortical sections (Fig. 1B), RORb expression in layer 4 on sagittal sections is high at the M/S1 and the S1/V1 borders. In cKO cortices, the RORb expression delineating the M/S1 border aligns with the border in WT brains, but the S1/V1 border shifts rostrally. In addition to layer 4, Cad8 also labels layers 2–3: Cad8 expression is high in layers 2–4 in M, low in S1, and high in V1 layers 2–4, revealing area borders. Overall in cKO cortices the Cad8 expression borders remain sharp, but the S1/V1 border shifts rostrally, whereas the M/S1 border remains unchanged. Area-specific layer 5 markers, such as ER81 (ETS-related protein 81) and SMI32 (Sternberger Zembrzycki et al.

Fig. 2. Concomitant shifts of molecular markers and input/output connections across layers in cKO cortices. (A) ISH on P7 sagittal sections shows the borders of area-specific Cad8 (layers 2–4), RORb (layer 4), and ER81 and SMI32 (both layer 5) expression (red arrowhead: M/S1 border; black arrowheads: S1/V1 border and caudal V1 border). The M/S1 border aligns in WT and cKO sections, but the S1/V1 border shifts rostrally in the cKO cortex. (B) 5HT staining on P7 sagittal sections shows TCA input into layer 4, revealing a rostral shift of the S1/V1 border in cKO. Retrograde labeling of corticospinal (DiI inserted into the CST) and corticotectal (DiD inserted into the SC) layer 5 output projections in P14 brains also reveals a rostrally shifted S1/V1 border (red arrowheads) in cKO sections. (Scale bars: 0.5 mm.)

monoclonals neurofilament 32) (high in M and S1, low in V1), also showed a rostral shift of the S1/V1 border in cKO sections. In upper and lower cortical layers, specific markers delineating the M/S1 border are maintained, but markers highlighting the S1/V1 border shift rostrally in cKO sections. We next tested whether, in addition to the altered subcortical input projections from the principal thalamic sensory nuclei into cortical layer 4 (5HT staining in Figs. 1 and 2B), layer 5 cortical output projections also could be affected in cKO. In P14 brains (Fig. 2B), corticospinal layer 5 projections in M/S1 were retrogradely labeled by inserting DiI crystals into the corticospinal tract (CST), whereas corticotectal layer 5 projections from V1 were labeled by inserting DiD crystals into the superior colliculus (SC). In WT and cKO sagittal sections, the two dyes labeled nonoverlapping neuronal populations, revealing a separation of corticospinal versus corticotectal output projections in layer 5 that predicts the S1/V1 border (Fig. 2B). In accordance with the expansion of molecular V1 markers and subcortical input from the dLG into V1 layer 4, corticotectal output projections in layer 5 also expand rostrally at the expense of corticospinal projections in cKO brains (Fig. 2B). This analysis shows that deletion of Lhx2 in postmitotic neurons broadly alters neuronal properties that affect area patterning across layers in the CP. Lhx2 Controls Area Patterning Specifically in Postmitotic Neurons. To analyze if postmitotic deletion of Lhx2 affects area patterning indirectly by retrogradely influencing the activity of area-patterning regulators in the VZ, we performed ISH for the TFs Emx2 (empty spiracles homeobox 2) (14-16), Pax6 (paired box 6) (14, 17, 18), CoupTF1 (chicken ovalbumin upstream promoter transcription factor) (1, 19), and Sp8 (specificity protein 8) immunolabelings (20, 21) on E12 sagittal sections. The expression of TFs remained Zembrzycki et al.

Lhx2 Controls Different Classes of Downstream Intermediate Regulators.

We next aimed to explore the mechanisms by which Lhx2 controls neuronal properties. However, compared with mechanisms in progenitors, relatively little is known about mechanisms that influence area-specific properties in postmitotic neurons (2). The few studied intermediate genetic regulatory elements (23) that influence aspects of area patterning are the TFs Bhlhb5 (basic helix-loop-helix domain containing class B5), Lmo4 (LIM domain only 4), and CoupTF1. Loss of Bhlhb5 function interferes with the acquisition of sensory area features in select cortical cell types and results in a disorganized S1 and altered corticospinal projections (23). Similarly, sustained Lmo4 function is required to maintain proper S1 shape and ordered segregation of TCAs in S1 (24-26). In neurons, CoupTF1 regulates postmitotic aspects of area patterning through a feedback mechanism with VZ patterning genes (27). To elucidate whether Lhx2 could affect the genetic profile in cortical neurons by regulating the activity of intermediate area-patterning regulators, we performed ISH and immunohistochemistry (IHC) for Bhlhb5, Lmo4, and CoupTF1 on P7 sagittal sections (Fig. 4A) as examples for intermediate regulators with known postmitotic functions (2). High Bhlhb5 immunolabeling in WT sections is evident in S1 but is much lower in M and V1. Expression of Lmo4 in the CP is complementary to Bhlhb5 (2), with strong expression in M/V1 and much lower activity in S1. CoupTF1 is strongly expressed in layer 4 across areas and at much lower levels in layers 2–3 in M and V1. In cKO sections, Bhlhb5 and Lmo4 are expressed at very low levels, whereas CoupTF1 expression conversely appears up-regulated, compared with WT sections (Fig. 4A). Lhx2 activity in neurons is required for normal expression levels of downstream TFs. Normal expression of these intermediate area-patterning regulators is required to drive area-specific genetic profiles in postmitotic cortical neurons (2, 23–27). In addition to direct transcriptional regulation, epigenetic mechanisms also impact on gene expression in many cell types, including neurons, by controlling DNA methylation (28, 29). Recent studies show that epigenetic mechanisms impact on area-specific gene expression in V1 in primates (30) and are needed for the normal development of S1 in mice (31, 32). To investigate if Lhx2 function could regulate epigenetic mechanisms, we analyzed the expression of candidate epigenetic regulators using immunostaining on P7 sagittal sections (Fig. 4B). We found that variations in the expression levels of the methyl–CpG–binding protein MeCP2 (high in V1 layers 2–3 and S1 layer 4, lower in M) (Fig. 4B) and the histone deacetylase HDAC1 (strong in V1 layer 2 PNAS Early Edition | 3 of 6

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unchanged in the VZ in cKO sections (Fig. 3A). Also at E14, a stage after area specification in the VZ (1–3), Tbr2, Pax6, CoupTF1, and SP8 expression remains unchanged in cKO (Fig. 3B) indicating that cortical progenitors have normal area identities in cKO embryos. Normal expression of TFs that are critical for cortical neurogenesis, such as Pax6 and Tbr2 (13, 22), also indicates that the molecular specification and generation of the different cortical layer neurons does not require postmitotic activity of Lhx2. In fact, we analyzed this issue at P7 by using ISH for the specific cortical layer markers Cux2 (Cut-like homeobox 2, layer 2–3), RORb (layer 4), ER81 (layer 5), and Tbr1 (layer 6) and found that cortical layers appear indistinguishable among genotypes (Fig. 3C). We conclude that the activity of Lhx2 in postmitotic neurons is dispensable for corticogenesis and layer formation but is critically required to sustain major hallmarks determining cortical area patterning (2, 3, 18) including area-specific genetic profiles and areaspecific cortical input/output projections. Despite its normal specification in progenitors, area identity is altered in the CP of cKO postmitotically, revealing that the transfer of area identity from progenitors to daughter neurons is malleable and that as a consequence the area information in neurons is misaligned. Sustained Lhx2 function in neurons is required to maintain normal area patterning in cortical neurons.

Fig. 3. Progenitor area identity and layer formation are unaffected in cKO cortices. (A) ISH using probes for Emx2, Pax6, CoupTF1, and Sp8 immunolabeling on E12 sagittal sections reveals indistinguishable expression gradients of patterning TFs in the cortical VZ (1) among genotypes. (B) ISH for Pax6, CoupTF1, and Sp8 immunolabeling at E14 also reveals comparable marker gene expression in WT and cKO sections. Pax6-stained sections then were double-labeled with a Tbr2 antibody. Among genotypes, both markers are expressed at comparable levels (shown in the high-magnification image) in the VZ (Pax6) and SVZ (Tbr2). (C) Nissl staining and ISH for the layerspecific marker genes Cux2 (layers 2–3), RORb (layer 4), ER81 (layer 5), and Tbr1 (layer 6) on P7 coronal sections reveals normal lamination in cKO cortices. vTEL, ventral telencephalon. (Scale bars: 0.5 mm.)

and S1 layers 2–3, very strong in S1 barrels) (Fig. 4B) is arearelated and allows the delineation of area borders in the CP (arrowheads in Fig. 4B). MeCP2 and HDAC1 influence DNA methylation (28, 33), and their expression pattern (Fig. 4B) suggests that they could operate in an area-specific fashion in the CP. Therefore we speculated that the distribution of epigenetic marks (e.g., 5-methylcytosine, 5Mc) that reflect chemical modifications of the DNA (28, 33) also could vary between areas. Consistent with the differences in MeCP2 and HDAC1 staining, which approximate cortical areas (arrowheads in Fig. 4B), 5Mc immunostaining also appears variable in the WT CP (arrowheads in Fig. 4B: high in V1 upper layers and in S1 layer 4), revealing the V1/S1 border. We next investigated MeCP2, HDAC1, and 5Mc staining in cKO sections and found that their staining intensities overall appear lower and more uniform than in WT cortices (asterisks in Fig. 4B). This analysis demonstrates that the differential distribution of epigenetic regulators (e.g., MeCP2) and their associated enzymes (e.g., HDAC1) among cortical regions matches the differential distribution of their substrates (e.g., 5Mc) in CP neurons in normal (WT) and abnormal (cKO) conditions, suggesting that regional differences in DNA methylation (28, 29, 33) could parallel (Figs. 1, 2, and 4A) and predict (Fig. 4B) gene expression differences in cKO cortical areas. To test this hypothesis, we isolated P7 genomic DNA from frontal, parietal, and occipital cortex and analyzed DNA methylation by using an ELISA for 5Mc. Quantification revealed (Fig. 4C) that the abundance of 5Mc in WT samples, measured as a percent of the total 4 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.1424440112

genomic DNA, differs significantly between areas (frontal/parietal cortex, P < 0.0001; parietal/occipital cortex, P = 0.0002). The highest percentages of 5Mc are evident in frontal cortex (1.36 ± 0.037%), the lowest in parietal cortex (1.02 ± 0.068%), and intermediate in occipital cortex (1.24 ± 0.024%). With 1.20 ± 0.051% in frontal cortex, 1.15 ± 0.044% in parietal cortex, and 1.09 ± 0.047% in occipital cortex, the quantification revealed significantly altered 5Mc abundance among the tested cortical areas in cKO samples (WT cKO in frontal cortex: P = 0.0016; parietal cortex: P = 0.0055; occipital cortex: P = 0.0002). Abnormally expressed epigenetic regulators and altered total DNA methylation in cKO, together with area abnormalities in related models (31, 32), suggest that epigenetic mechanisms could complement the established role of intermediate TFs (2) in regulating area properties. Next, to test whether the abnormal expression of downstream regulators is evident only after area features are differentiated (e.g., P7) or appears before these features emerge, we analyzed P0 sagittal sections. We found that all markers that are affected in cKO cortices at P7 (Fig. 4 and Fig. S8) already appear abnormal in cKO sections at P0 (Fig. S9). For example, the opposing down-regulation of Bhlhb5/Lmo4 and up-regulation of CoupTF1 already is evident in cKO sections at P0, although less pronounced than at later times (compare Fig. 4 A and B and Fig. S9). Lhx2 progressively controls the activity of its downstream regulators, starting before sensory areas emerge. Mediated through its DNA-binding homeodomain, Lhx2 controls hundreds of downstream genes directly (34, 35). We next aimed to identify molecules that are relevant to cortical area patterning and, mediated by its protein-binding LIM domain (36), interact biochemically with Lhx2. We performed immunoprecipitation (IP) and coimmunoprecipitation (co-IP) assays for molecules that are abnormally expressed in cKO brains (Fig. 4 and Fig. S9). IP assays revealed proteins of the TFs Lhx2, CoupTF1, and Bhlhb5 and the epigenetic regulators MeCP2 and HDAC1 in P0 cortical lysates (Fig. 4D). Using co-IPs, we then found that Lhx2 does not bind directly to CoupTF1 and MeCP2 but binds to Bhlhb5 and HDAC1 protein in vivo (Fig. 4 E and F). These experiments identify two novel Lhx2interacting proteins. Further, Lhx2 interacts physically with some but not all of its downstream effectors that show affected expression in cKO cortices. We conclude that Lhx2 function in neurons is critical for the establishment of normal sensory area patterning in the CP, presumably by regulating parallel postmitotic mechanisms that drive the establishment of principal properties of cortical neurons. Discussion How, after their initial specification in progenitors, cortical area properties are set up and maintained in cortical neurons remained largely elusive. Our findings imply major revelations to existing models describing area patterning (1–3) by demonstrating that specification of area identity in progenitors is insufficient. Using conditional inactivation of Lhx2 in postmitotic cortical neurons, we show that hallmarks describing area patterning (1–3), including (i) complementary gene expression across layers in the CP, (ii) the targeting of subcortical input projections from the principal sensory thalamic nuclei into the cortex, and (iii) the balance between corticospinal and corticotectal output projections, are concomitantly altered in brains that specifically lack Lhx2 function in postmitotic cortical neurons (see schematic in Fig. S10). This postmitotic function of Lhx2 in cortical neurons regulates area patterning after patterning TFs already have prespecified these features in cortical progenitors (1–3). This observation is very interesting, because it demonstrates that the current view about the hierarchy of molecular events that determine area identity in the cortex is not accurate. In the existing model, patterning in progenitors strictly dictates the area identity of the neurons that they generate (1–3). We suggest a more comprehensive model of area patterning that defines the Zembrzycki et al.

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Fig. 4. Downstream regulators are misexpressed in cKO neurons. (A) ISH on P7 sagittal sections shows complementary expression patterns of Bhlhb5 and Lmo4 (Bhlhb5: low in M and V1, strong in S1; Lmo4: strong in M and V1, low in S1) in the cortex. Overall, the expression of both markers is strongly downregulated in cKO cortices. CoupTF1 ISH reveals strong expression in layer 4 across areas and in layers 2–3 in V1 and M. CoupTF1 expression appears strongly upregulated in the cKO cortex as compared with the WT cortex. (B) Immunostaining on P7 sagittal WT sections for MeCP2 (high in V1 layers 2–3 and S1 layer 4), HDAC1 (high in V1 layer 2 and in S1 layers 2–3, very strong in S1 barrels), and 5Mc (high in V1 layers 2–3 and S1 layer 4) is broad overall, but differences in staining intensities approximate area borders (arrowheads) in the CP. In cKO, these markers remain expressed but at lower levels and more uniformly in the CP (asterisks). (C) The content of 5Mc in total DNA samples derived from P7 frontal, parietal, and occipital cortices was measured using an ELISA (5Mc content is displayed as percent of total DNA). In WT samples, the 5Mc content is highest in frontal (1.36 ± 0.037%), lowest in parietal (1.02 ± 0.068%), and intermediate in occipital cortex (1.24 ± 0.024%). With 1.20 ± 0.051% (P = 0.0016) in frontal, 1.15 ± 0.044% (P = 0.0055) in parietal, and 1.09 ± 0.047% (P = 0.0002) in occipital cortex, 5Mc percentages in cKO areas differ significantly from the percentages in WT areas. *P < 0.05. (D) IP reveals that Lhx2, Bhlhb5, CoupTF1, MeCP2, and HDAC1 proteins are abundant in P0 cortical lysates in vivo. (E and F) Lhx2 does not coimmunoprecipitate with CoupTF1 or MeCP2 protein. Conversely, Bhlhb5 and HDAC1 proteins do coimmunoprecipitate with Lhx2 in P0 cortical lysates in vivo. Numbers in D–F indicate protein size markers in kilodaltons. (Scale bars: 0.5mm.)

previously unknown intermediate processes in postmitotic cortical neurons. These processes include the role of Lhx2 in the regulation of postmitotic area identity that dominates the area features determined in VZ progenitors: After the initial specification of sensory area blueprints driven by graded expression of patterning TFs in the VZ progenitors (1–3), we propose that the sustained activity of key alignment TFs, including Lhx2, in neurons is a crucial requirement to linearly transfer and coordinate area identity in daughter neurons (Fig. S10). Current knowledge suggests that intermediate regulators execute the acquisition of area features in neurons. Each of them conveys select properties in specific cell types, layers, or areas (2). Our findings are in line with this view and in addition support an emerging concept in the field (37) which suggests that epigenetic mechanisms contribute to this process. We propose that epigenetic mechanisms should be considered as additional intermediate regulators of sensory area features in cortical neurons. Our experiments suggest at least two different classes of intermediate regulators that influence sensory area properties in postmitotic cortical neurons and a hierarchy between them. The first class, classic intermediate genetic regulatory elements, reflect diverse types of molecules including TFs such as Bhlhb5, Lmo4, and CoupTF1 (2, 23, 25–27), epigenetic regulators such as MeCP2 and HDAC1 (29–31, 37, 38), and likely others that affect select features of sensory areas and/or layers without driving overall area identity across cortical layers and areas. The second class are terminal selector genes, including Lhx2, that operate Zembrzycki et al.

upstream of intermediate regulators. Importantly, terminal selector genes impact area patterning substantially by controlling the entire array of features concomitantly, across areas and layers in the CP postmitotically. In invertebrate and vertebrate species, terminal selector genes, including Lhx2 homologs, establish the definitive properties in many cell types by driving cell type-specific batteries of terminal differentiation genes (39–41). Our study using mice complements these findings by showing that postmitotic Lhx2 function controls the area-specific terminal differentiation program in cortical neurons. Interestingly, we also have identified Bhlhb5 and HDAC1 as two novel Lhx2-interacting proteins. LIM-homeodomain TFs such as Lhx2 are well known to affect cellular processes through binding to other proteins (36). Future studies that detail the cellular and molecular consequences of Lhx2 in association with its interacting proteins are needed to understand how it affects neuronal properties in parallel with its role as a powerful transcriptional regulator (34, 35). Our study addresses key unanswered issues about how sensory area features are specified in postmitotic neurons. A recent study (27) reports that postmitotic deletion of CoupTF1 results in area patterning abnormalities that are reminiscent to its deletion in progenitors (19). This study fails to reveal exclusive area-patterning mechanisms in postmitotic neurons. Postmitotic functions of CoupTF1 influence the activity of patterning TFs in the VZ through a feedback mechanism, which in turn seems to drive these defects (27). Conversely, after postmitotic deletion of Lhx2, area patterning in progenitors remains normal. This result PNAS Early Edition | 5 of 6

clearly demonstrates that Lhx2 is required to regulate sensory area properties by controlling all hallmarks of arealization specifically in postmitotic cortical neurons. In summary, our findings add novel insights into the sequence and hierarchy of mechanisms that regulate the layout of the cortex into sensory areas postmitotically. The revised model has major implications for the current understanding and for the further investigation of the complex neurodevelopmental mechanisms that establish proper sensory circuits and their functions.

conducted following the guidelines of the Institutional Animal Care and Use Committee at the Salk Institute and were in full compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (42). Staining. ISH, immunostaining, and axon tracings were performed as described in refs. 4, 18, and 21. The detailed protocols for DNA methylation analysis and IPs can be found in SI Materials and Methods along with the description of reported measurements and statistical analyses.

Mouse Lines. The mouse lines used in this study along with their sources are described in SI Materials and Methods. All experiments were approved and

ACKNOWLEDGMENTS. We thank Klaus A. Nave for providing Nex-Cre mice and Tom Jessell (Lhx2), Robert Hevner (Tbr1), and Sarah E. Ross (Bhlhb5) for providing antibodies. This work was supported by National Institutes of Health Grants R37 NS31558 and R01 MH086147 (to D.D.M.O.) and National Science Council, Taiwan Grant 102-2321-B-001-035 (to S.-J.C.). D.D.M.O. holds the Vincent J. Coates Chair of Molecular Neurobiology at the Salk Institute for Biological Sciences.

1. O’Leary DDM, Stocker AM, Zembrzycki A (2013) Area patterning of the mammalian cortex. Patterning and Cell Type Specification in the Developing Cns and Pns, Chapter 4, eds John R, Pasko R (Academic, Oxford, UK), pp 61–85. 2. Greig LC, Woodworth MB, Galazo MJ, Padmanabhan H, Macklis JD (2013) Molecular logic of neocortical projection neuron specification, development and diversity. Nat Rev Neurosci 14(11):755–769. 3. Rakic P (2009) Evolution of the neocortex: A perspective from developmental biology. Nat Rev Neurosci 10(10):724–735. 4. Chou SJ, Perez-Garcia CG, Kroll TT, O’Leary DD (2009) Lhx2 specifies regional fate in Emx1 lineage of telencephalic progenitors generating cerebral cortex. Nat Neurosci 12(11):1381–1389. 5. Mangale VS, et al. (2008) Lhx2 selector activity specifies cortical identity and suppresses hippocampal organizer fate. Science 319(5861):304–309. 6. Chou SJ, O’Leary DD (2013) Role for Lhx2 in corticogenesis through regulation of progenitor differentiation. Mol Cell Neurosci 56:1–9. 7. Shetty AS, et al. (2013) Lhx2 regulates a cortex-specific mechanism for barrel formation. Proc Natl Acad Sci USA 110(50):E4913–E4921. 8. Roy A, Gonzalez-Gomez M, Pierani A, Meyer G, Tole S (2014) Lhx2 regulates the development of the forebrain hem system. Cereb Cortex 24(5):1361–1372. 9. Goebbels S, et al. (2006) Genetic targeting of principal neurons in neocortex and hippocampus of NEX-Cre mice. Genesis 44(12):611–621. 10. D’Amato RJ, et al. (1987) Ontogeny of the serotonergic projection to rat neocortex: Transient expression of a dense innervation to primary sensory areas. Proc Natl Acad Sci USA 84(12):4322–4326. 11. Fujimiya M, Kimura H, Maeda T (1986) Postnatal development of serotonin nerve fibers in the somatosensory cortex of mice studied by immunohistochemistry. J Comp Neurol 246(2):191–201. 12. Rhoades RW, et al. (1990) Development and lesion induced reorganization of the cortical representation of the rat’s body surface as revealed by immunocytochemistry for serotonin. J Comp Neurol 293(2):190–207. 13. Sun T, Hevner RF (2014) Growth and folding of the mammalian cerebral cortex: From molecules to malformations. Nat Rev Neurosci 15(4):217–232. 14. Bishop KM, Goudreau G, O’Leary DD (2000) Regulation of area identity in the mammalian neocortex by Emx2 and Pax6. Science 288(5464):344–349. 15. Hamasaki T, Leingärtner A, Ringstedt T, O’Leary DD (2004) EMX2 regulates sizes and positioning of the primary sensory and motor areas in neocortex by direct specification of cortical progenitors. Neuron 43(3):359–372. 16. Leingärtner A, et al. (2007) Cortical area size dictates performance at modalityspecific behaviors. Proc Natl Acad Sci USA 104(10):4153–4158. 17. Piñon MC, Tuoc TC, Ashery-Padan R, Molnár Z, Stoykova A (2008) Altered molecular regionalization and normal thalamocortical connections in cortex-specific Pax6 knock-out mice. J Neurosci 28(35):8724–8734. 18. Zembrzycki A, Chou SJ, Ashery-Padan R, Stoykova A, O’Leary DD (2013) Sensory cortex limits cortical maps and drives top-down plasticity in thalamocortical circuits. Nat Neurosci 16(8):1060–1067. 19. Armentano M, et al. (2007) COUP-TFI regulates the balance of cortical patterning between frontal/motor and sensory areas. Nat Neurosci 10(10):1277–1286. 20. Sahara S, Kawakami Y, Izpisua Belmonte JC, O’Leary DD (2007) Sp8 exhibits reciprocal induction with Fgf8 but has an opposing effect on anterior-posterior cortical area patterning. Neural Dev 2:10.

21. Zembrzycki A, Griesel G, Stoykova A, Mansouri A (2007) Genetic interplay between the transcription factors Sp8 and Emx2 in the patterning of the forebrain. Neural Dev 2:8. 22. Elsen GE, et al. (2013) The protomap is propagated to cortical plate neurons through an Eomes-dependent intermediate map. Proc Natl Acad Sci USA 110(10):4081–4086. 23. Joshi PS, et al. (2008) Bhlhb5 regulates the postmitotic acquisition of area identities in layers II-V of the developing neocortex. Neuron 60(2):258–272. 24. Kashani AH, et al. (2006) Calcium activation of the LMO4 transcription complex and its role in the patterning of thalamocortical connections. J Neurosci 26(32):8398–8408. 25. Huang Z, et al. (2009) Transcription factor Lmo4 defines the shape of functional areas in developing cortices and regulates sensorimotor control. Dev Biol 327(1):132–142. 26. Cederquist GY, Azim E, Shnider SJ, Padmanabhan H, Macklis JD (2013) Lmo4 establishes rostral motor cortex projection neuron subtype diversity. J Neurosci 33(15):6321–6332. 27. Alfano C, Magrinelli E, Harb K, Hevner RF, Studer M (2014) Postmitotic control of sensory area specification during neocortical development. Nat Commun 5:5632. 28. Jones PL, et al. (1998) Methylated DNA and MeCP2 recruit histone deacetylase to repress transcription. Nat Genet 19(2):187–191. 29. Lister R, et al. (2013) Global epigenomic reconfiguration during mammalian brain development. Science 341(6146):1237905. 30. Hata K, et al. (2013) DNA methylation and methyl-binding proteins control differential gene expression in distinct cortical areas of macaque monkey. J Neurosci 33(50): 19704–19714. 31. Golshani P, Hutnick L, Schweizer F, Fan G (2005) Conditional Dnmt1 deletion in dorsal forebrain disrupts development of somatosensory barrel cortex and thalamocortical long-term potentiation. Thalamus Relat Syst 3(3):227–233. 32. Moroto M, et al. (2013) Altered somatosensory barrel cortex refinement in the developing brain of Mecp2-null mice. Brain Res 1537:319–326. 33. Jones PA, Takai D (2001) The role of DNA methylation in mammalian epigenetics. Science 293(5532):1068–1070. 34. Hou PS, et al. (2013) LHX2 regulates the neural differentiation of human embryonic stem cells via transcriptional modulation of PAX6 and CER1. Nucleic Acids Res 41(16): 7753–7770. 35. Mardaryev AN, et al. (2011) Lhx2 differentially regulates Sox9, Tcf4 and Lgr5 in hair follicle stem cells to promote epidermal regeneration after injury. Development 138(22):4843–4852. 36. Kadrmas JL, Beckerle MC (2004) The LIM domain: From the cytoskeleton to the nucleus. Nat Rev Mol Cell Biol 5(11):920–931. 37. Krubitzer L, Stolzenberg DS (2014) The evolutionary masquerade: Genetic and epigenetic contributions to the neocortex. Curr Opin Neurobiol 24(1):157–165. 38. Fagiolini M, Jensen CL, Champagne FA (2009) Epigenetic influences on brain development and plasticity. Curr Opin Neurobiol 19(2):207–212. 39. Hobert O (2008) Regulatory logic of neuronal diversity: Terminal selector genes and selector motifs. Proc Natl Acad Sci USA 105(51):20067–20071. 40. Hobert O (2011) Regulation of terminal differentiation programs in the nervous system. Annu Rev Cell Dev Biol 27:681–696. 41. Zhang F, et al. (2014) The LIM and POU homeobox genes ttx-3 and unc-86 act as terminal selectors in distinct cholinergic and serotonergic neuron types. Development 141(2):422–435. 42. Committee on Care and Use of Laboratory Animals (1996) Guide for the Care and Use of Laboratory Animals (Natl Inst Health, Bethesda), DHHS Publ No (NIH) 85-23.

Materials and Methods

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