Supporting Information - PNAS · PFA, postfixed in Bouin’s solution (Sigma), dehydrated with...
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Supporting Information Zembrzycki et al. 10.1073/pnas.1424440112 SI Materials and Methods Mouse Lines. All experiments were approved and conducted fol- lowing the guidelines of the Institutional Animal Care and Use Committee at the Salk Institute and were in full compliance with the guidelines of the National Institutes of Health for the care and use of laboratory animals. When mice were mated, the morning of the identified vaginal plug was designated as E0.5. The morning on which pups were born was designated P 0.5. To generate mice with a selective deletion of Lhx2 in postmitotic neurons (herein termed “cKO”), we crossed Lhx2 floxed (Lhx2 fl/fl ) mice (1) with Nex-Cre–expressing mice (2). To assess specificity of Cre- recombination in mouse embryos, mice expressing the fluorescent reporter AI14-tdTomato (3) [Jackson Laboratory: B6;129S6-Gt (ROSA)26Sortm14(CAT-tdTomato)Hze/J] were crossed to mice expressing Nex-Cre. Genotyping was done by PCR as previously described (1, 2). ISH, IHC, and Tangential Cortical Sections. Embryos were dissected, washed in PBS, fixed overnight in 4% (wt/vol) phosphate-buffered paraformaldehyde (PFA), washed in PBS, and cryopreserved in 30% (wt/vol) sucrose in PBS. Postnatal brains were perfused with PFA, postfixed overnight in PFA, washed with PBS, and cry- opreserved in 30% (wt/vol) sucrose in PBS. Tissues were em- bedded in Tissue-Tek OCT (Sakura Finetek) and sectioned on a cryostat (Leica). Antisense RNA probes for ISH were labeled using a DIG-RNA labeling kit (Roche). ISH and immunostain- ings on 18-μm cryostat sections were carried out as previously described (1, 4). For IHC, sections were fixed in 2% (wt/vol) PFA, postfixed in Bouin’s solution (Sigma), dehydrated with graded alcohols, embedded in paraffin, and cut in a microtome into 10-μm slices (1). For tangential cortical sections, cortical hemispheres were dissected, flattened, postfixed between slide glasses, and then cryoprotected. Tangential sections were cut into 40-μm slices from flattened cortical hemispheres with a sliding microtome and then were immunostained for 5HT or were mounted on slides and used for ISH. For Nissl staining, sections were stained with 0.5% cresyl violet and then dehydrated with graded alcohols. IHC was performed on 10- to 18-μm coronal or sagittal or 40-μm tangential cryosections or paraffin sections. An- tigens were unmasked by boiling in citrate buffer (Vector). Primary antibodies used were rabbit anti-5HT (1:20,000; ImmunoStar), rabbit anti-SMI32 (1:5,000; Covance); rabbit anti-MeCP2 (1:1,000; Cell Signaling); rabbit anti-5Mc (1:1,000; Epigentek); rabbit anti- HDAC1 (1:2,000; Millipore); rabbit anti-Lhx2 (1:1,000; gift from Tom Jessell, Columbia University, New York, NY); goat anti-Lhx2 (1:500; Santa Cruz); rabbit anti-Tbr2 (1:1,000; Abcam); mouse anti- Tuj1 (1:2,000; Covance); rabbit anti-Sp8 (1:20,000; Millipore); rabbit anti-Tbr1 (1:1,000; gift from Robert Hevner, University of Washington, Seattle, WA); rabbit anti-Ctip2 (1:1,000; Novus Pharmaceuticals); mouse anti-Satb2 (1:1,000; Abcam); and rabbit anti-Bhlhb5 (1:1,000; gift from Sarah E. Ross, University of Pittsburgh, Pittsburgh, PA). Primary antibodies were incubated overnight at 4 °C in blocking solution containing 3% (wt/vol) BSA (Fisher Scientific) and 0.1% Tween 20 (Sigma) in PBS. Bio- tinylated (Jackson ImmunoResearch) or Alexa-conjugated (Mo- lecular Probes) secondary antibodies were incubated for 45 min at room temperature and then were washed with PBS. Cell nuclei were counterstained using DAPI (Vector). Nonfluorescent im- munostaining was developed using the diaminobenzidine colori- metric reaction and the Vectastain kit (Vector). Axon Tracings. Lipophilic tracers DiI, DiO, and DiD (all from Molecular Probes) were used to label corticothalamic, cortico- tectal, and corticospinal projections. In both WT and cKO, small crystals of the carbocyanide dyes were inserted into comparable stereotaxic locations corresponding to S1 and V1 of P7 brains to label thalamocortical connections; in P14 brains dyes were inserted into the SC and CST to label corticotectal and cortico- spinal projections, respectively. Brains were incubated for 4–16 wk in 4% (wt/vol) PFA and then were embedded in 5% (wt/vol) low- melting point agarose, cut into 100-μm-thick coronal or sagittal sections on a vibratome (Leica), counterstained with DAPI (Vector), and photographed under fluorescent light. For each experiment six to eight brains with comparable tracer injection sites were cut and used for further data analysis. Representative example images are shown in the figures. DNA Methylation Analysis. To measure the content of 5-hydroxy- methylated cytosine in different cortical areas, unfixed P7 cortices were dissected, isolated from subcortical tissue, and cut into fragments corresponding to the frontal, parietal, and occipital cortex. Genomic DNA from these tissue samples was extracted by using the GenElute Mammalian Genomic DNA Miniprep Kit (Sigma). One hundred nanograms of genomic DNA per sample were used to perform an ELISA designed specifically to detect 5-hydroxymethylated cytosine using the colorimetric MethylFlash Methylated DNA Quantification Kit (P-1034; Epigentek) and a fluorescent plate reader (HTS 7000; Perkin-Elmer). Detected fluorescence of the ELISA samples was used to calculate content and percentages of 5Mc using the manufacturer’s protocol (Epi- gentek) and equation (5). Three different brain samples were used for each tested brain region and genotype. Each resulting ELISA sample was analyzed and measured in triplicate. Final quantifi- cations show the mean values of detected 5Mc in WT and cKO samples as percent of total DNA and indicate the SEM. IP. Cortices of newborn mice were dissected and lysed in RIPA buffer (150 mM NaCl, 1% Nonidet P-40, 0.5% DOC, 0.1% SDS, 50 mM Tris, pH 8.0), and the concentration of protein present in the lysates was measured using Bradford’s assay. IP and co-IP then were performed as described previously (6) using the Lhx2, Bhlhb5, CoupTF1, MeCP2, and HDAC1 antibodies detailed in the above section. After a preclear step, protein G Sepharose beads (GE Healthcare, Invitrogen) were incubated at 4 °C overnight with the IP antibody and the protein sample. Samples were further washed, resuspended in standard Western blot sample buffer, boiled, and resolved in SDS/PAGE. For co-IP, blots were further immunoblotted with an antibody against the potential interacting protein. Blots were developed using a standard chemoluminescence assays (Amersham Pharmacia, Roche). Cortical Area Measurements and Statistical Analysis. Neocortical surface area and size of cortical primary sensory areas was measured on 40-μm horizontal sections stained with 5HT antibody. For each brain analyzed, area measurements were made on composite aligned and collapsed sections that showed 5HT staining by using ImageJ (7). Quantifications in Fig. 1 show all individual data points and mean values of the tested specimens. Analyzed numbers of samples for each set of experiments are indicated in the figures and figure legends. Statistical significance was determined using un- paired two-tailed t test, P values < 0.05 (indicated as *) were con- sidered as statistically significant. Variance is indicated in the text and corresponding figure legends reflecting SEM. Zembrzycki et al. www.pnas.org/cgi/content/short/1424440112 1 of 7
Transcript of Supporting Information - PNAS · PFA, postfixed in Bouin’s solution (Sigma), dehydrated with...
Supporting InformationZembrzycki et al. 10.1073/pnas.1424440112SI Materials and MethodsMouse Lines. All experiments were approved and conducted fol-lowing the guidelines of the Institutional Animal Care and UseCommittee at the Salk Institute and were in full compliance withthe guidelines of the National Institutes of Health for the care anduse of laboratory animals. Whenmice were mated, the morning ofthe identified vaginal plug was designated as E0.5. The morningon which pups were born was designated P 0.5. To generate micewith a selective deletion of Lhx2 in postmitotic neurons (hereintermed “cKO”), we crossed Lhx2 floxed (Lhx2fl/fl) mice (1)with Nex-Cre–expressing mice (2). To assess specificity of Cre-recombination in mouse embryos, mice expressing the fluorescentreporter AI14-tdTomato (3) [Jackson Laboratory: B6;129S6-Gt(ROSA)26Sortm14(CAT-tdTomato)Hze/J] were crossed to miceexpressing Nex-Cre. Genotyping was done by PCR as previouslydescribed (1, 2).
ISH, IHC, and Tangential Cortical Sections. Embryos were dissected,washed in PBS, fixed overnight in 4% (wt/vol) phosphate-bufferedparaformaldehyde (PFA), washed in PBS, and cryopreserved in30% (wt/vol) sucrose in PBS. Postnatal brains were perfused withPFA, postfixed overnight in PFA, washed with PBS, and cry-opreserved in 30% (wt/vol) sucrose in PBS. Tissues were em-bedded in Tissue-Tek OCT (Sakura Finetek) and sectioned on acryostat (Leica). Antisense RNA probes for ISH were labeledusing a DIG-RNA labeling kit (Roche). ISH and immunostain-ings on 18-μm cryostat sections were carried out as previouslydescribed (1, 4). For IHC, sections were fixed in 2% (wt/vol)PFA, postfixed in Bouin’s solution (Sigma), dehydrated withgraded alcohols, embedded in paraffin, and cut in a microtomeinto 10-μm slices (1). For tangential cortical sections, corticalhemispheres were dissected, flattened, postfixed between slideglasses, and then cryoprotected. Tangential sections were cut into40-μm slices from flattened cortical hemispheres with a slidingmicrotome and then were immunostained for 5HT or weremounted on slides and used for ISH. For Nissl staining, sectionswere stained with 0.5% cresyl violet and then dehydrated withgraded alcohols. IHC was performed on 10- to 18-μm coronal orsagittal or 40-μm tangential cryosections or paraffin sections. An-tigens were unmasked by boiling in citrate buffer (Vector). Primaryantibodies used were rabbit anti-5HT (1:20,000; ImmunoStar),rabbit anti-SMI32 (1:5,000; Covance); rabbit anti-MeCP2 (1:1,000;Cell Signaling); rabbit anti-5Mc (1:1,000; Epigentek); rabbit anti-HDAC1 (1:2,000; Millipore); rabbit anti-Lhx2 (1:1,000; gift fromTom Jessell, Columbia University, New York, NY); goat anti-Lhx2(1:500; Santa Cruz); rabbit anti-Tbr2 (1:1,000; Abcam); mouse anti-Tuj1 (1:2,000; Covance); rabbit anti-Sp8 (1:20,000; Millipore);rabbit anti-Tbr1 (1:1,000; gift from Robert Hevner, Universityof Washington, Seattle, WA); rabbit anti-Ctip2 (1:1,000; NovusPharmaceuticals); mouse anti-Satb2 (1:1,000; Abcam); and rabbitanti-Bhlhb5 (1:1,000; gift from Sarah E. Ross, University ofPittsburgh, Pittsburgh, PA). Primary antibodies were incubatedovernight at 4 °C in blocking solution containing 3% (wt/vol)BSA (Fisher Scientific) and 0.1% Tween 20 (Sigma) in PBS. Bio-tinylated (Jackson ImmunoResearch) or Alexa-conjugated (Mo-lecular Probes) secondary antibodies were incubated for 45 min atroom temperature and then were washed with PBS. Cell nucleiwere counterstained using DAPI (Vector). Nonfluorescent im-munostaining was developed using the diaminobenzidine colori-metric reaction and the Vectastain kit (Vector).
Axon Tracings. Lipophilic tracers DiI, DiO, and DiD (all fromMolecular Probes) were used to label corticothalamic, cortico-tectal, and corticospinal projections. In both WT and cKO, smallcrystals of the carbocyanide dyes were inserted into comparablestereotaxic locations corresponding to S1 and V1 of P7 brainsto label thalamocortical connections; in P14 brains dyes wereinserted into the SC and CST to label corticotectal and cortico-spinal projections, respectively. Brains were incubated for 4–16 wkin 4% (wt/vol) PFA and then were embedded in 5% (wt/vol) low-melting point agarose, cut into 100-μm-thick coronal or sagittalsections on a vibratome (Leica), counterstained with DAPI(Vector), and photographed under fluorescent light. For eachexperiment six to eight brains with comparable tracer injectionsites were cut and used for further data analysis. Representativeexample images are shown in the figures.
DNA Methylation Analysis. To measure the content of 5-hydroxy-methylated cytosine in different cortical areas, unfixed P7 corticeswere dissected, isolated from subcortical tissue, and cut intofragments corresponding to the frontal, parietal, and occipitalcortex. Genomic DNA from these tissue samples was extracted byusing the GenElute Mammalian Genomic DNA Miniprep Kit(Sigma). One hundred nanograms of genomic DNA per samplewere used to perform an ELISA designed specifically to detect5-hydroxymethylated cytosine using the colorimetric MethylFlashMethylated DNA Quantification Kit (P-1034; Epigentek) and afluorescent plate reader (HTS 7000; Perkin-Elmer). Detectedfluorescence of the ELISA samples was used to calculate contentand percentages of 5Mc using the manufacturer’s protocol (Epi-gentek) and equation (5). Three different brain samples were usedfor each tested brain region and genotype. Each resulting ELISAsample was analyzed and measured in triplicate. Final quantifi-cations show the mean values of detected 5Mc in WT and cKOsamples as percent of total DNA and indicate the SEM.
IP. Cortices of newborn mice were dissected and lysed in RIPAbuffer (150 mM NaCl, 1% Nonidet P-40, 0.5% DOC, 0.1% SDS,50 mM Tris, pH 8.0), and the concentration of protein present inthe lysates was measured using Bradford’s assay. IP and co-IPthen were performed as described previously (6) using the Lhx2,Bhlhb5, CoupTF1, MeCP2, and HDAC1 antibodies detailed inthe above section. After a preclear step, protein G Sepharose beads(GE Healthcare, Invitrogen) were incubated at 4 °C overnight withthe IP antibody and the protein sample. Samples were furtherwashed, resuspended in standard Western blot sample buffer,boiled, and resolved in SDS/PAGE. For co-IP, blots were furtherimmunoblotted with an antibody against the potential interactingprotein. Blots were developed using a standard chemoluminescenceassays (Amersham Pharmacia, Roche).
Cortical Area Measurements and Statistical Analysis. Neocorticalsurface area and size of cortical primary sensory areas was measuredon 40-μm horizontal sections stained with 5HT antibody. For eachbrain analyzed, area measurements were made on compositealigned and collapsed sections that showed 5HT staining by usingImageJ (7). Quantifications in Fig. 1 show all individual data pointsand mean values of the tested specimens. Analyzed numbers ofsamples for each set of experiments are indicated in the figures andfigure legends. Statistical significance was determined using un-paired two-tailed t test, P values < 0.05 (indicated as *) were con-sidered as statistically significant. Variance is indicated in the textand corresponding figure legends reflecting SEM.
Zembrzycki et al. www.pnas.org/cgi/content/short/1424440112 1 of 7
1. Chou SJ, Perez-Garcia CG, Kroll TT, O’Leary DD (2009) Lhx2 specifies regional fate inEmx1 lineage of telencephalic progenitors generating cerebral cortex. Nat Neurosci12(11):1381–1389.
2. Goebbels S, et al. (2006) Genetic targeting of principal neurons in neocortex andhippocampus of NEX-Cre mice. Genesis 44(12):611–621.
3. Madisen L, et al. (2010) A robust and high-throughput Cre reporting and character-ization system for the whole mouse brain. Nat Neurosci 13(1):133–140.
4. Zembrzycki A, Chou SJ, Ashery-Padan R, Stoykova A, O’Leary DD (2013) Sensory cortexlimits cortical maps and drives top-down plasticity in thalamocortical circuits. NatNeurosci 16(8):1060–1067.
5. Li W, Liu M (2011) Distribution of 5-hydroxymethylcytosine in different human tissues.J Nucleic Acids 2011:870726.
6. Lim YS, et al. (2008) p75(NTR) mediates ephrin-A reverse signaling required for axonrepulsion and mapping. Neuron 59(5):746–758.
7. Rasband WS (1997-2014) ImageJ (US National Institutes of Health, Bethesda).
Fig. S1. Lhx2 is dynamically expressed in cortical progenitors and neurons in the CP. We investigated the time course of Lhx2 expression on coronal (A) andsagittal (B) sections using an antibody staining or ISH. (A) Lhx2 is expressed in a medial/high-to-lateral/low gradient in cortical VZ progenitors until around P0.In the intermediate zone (IZ) and developing CP, Lhx2 shows a lateral/high-to-medial/low expression countergradient that postnatally becomes more uniformand restricted to upper cortical layers. (B) Sagittal sections reveal that in progenitors and the CP until around P0, Lhx2 is expressed in a caudal/high-to-rostral/low gradient. By P7, Lhx2 becomes more uniformly expressed in upper cortical layers. 2–4, cortical layers 2–4; CTX, cortex; SVZ, subventricular zone. Main axes:A, anterior; D, dorsal; L, lateral.
Fig. S2. Nex-Cre conditionally deletes Lhx2 specifically in cortical postmitotic neurons. (A) To analyze the specificity of cre-recombination in the developingcortex, Nex-Cre mice were crossed to AI14 reporter mice that upon cre recombination express the fluorescent reporter tdTomato in recombined cells. On E12coronal sections of Nex-Cre/AI14 double-positive specimens, tdTomato reporter gene activity (red staining) reveals recombined cells only in the preplate (PP)and CP, respectively. Immunolabeling of Nex-Cre/AI14 double-positive specimens with the pan neuronal marker Tuj (green staining) confirms that tdTomato-expressing Nex-Cre recombined cells fully overlap with Tuj expression in the PP/CP. The VZ contains progenitors that do not express Tuj and also do not showtdTomato reporter gene activity. Sections were counterstained with the nuclear marker DAPI (blue staining). (B) Immunolabeling demonstrates robust Lhx2expression in the VZ/SVZ, IZ, and CP in WT E13 and E17 coronal sections. Immunostaining on sections derived from specimens that are homozygous for Lhx2floxed alleles and positive for Nex-Cre (conditional Lhx2 mutants, herein cKO) shows no Lhx2 staining in the IZ/CP. Lhx2 staining remains detectable at normallevels in the VZ/SVZ of cKO sections at both stages. (C) Dissected P7 WT and cKO whole-mount brains are indistinguishable in size and proportions. On P7coronal and sagittal sections Nissl staining was used to analyze brain neuroanatomy. Nissl staining revealed no differences in cortical size, cortical patterning,location of the piriform cortex/neocortex border (rhinal fissure; black pointer), or cortical lamination. BG, basal ganglia; CB, cerebellum; dTH, dorsal thalamus;HC, hippocampus; pCTX, piriform cortex; TH, thalamus; WM, white matter.
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Fig. S3. Subcortical connectivity is abnormal in cKO brains. Crystals of DiI (red dye in A and B) and DiO (green dye in A) or DiD (green dye in B) were inserted incomparable stereotaxic locations of P7 brains (arrowheads in whole-brain images in A and B indicate the injection sites). (A) Dyes were inserted in locationsthat in WT approximate the central domains of S1 (DiI) and V1 (DiO). DAPI-counterstained coronal WT and cKO sections at the level of the thalamus revealretrogradely DiI-labeled cells in the thalamic somatosensory nucleus (VPN), whereas retrogradely DiO-labeled cell bodies are evident in the visual thalamicnucleus (dLG; dashed outline). (B) Inserting DiI crystals into cortical areas that approximate the rostral V1 region and DiD (green dye) into regions that ap-proximate to the caudal S1 region in WT sections through the thalamus reveals a similar separation of retrogradely labeled cells in the VPN (red arrowheadsindicate DiI-labeled cells) and dLG (green arrowheads indicate DiD-labeled cells). Inserting these dyes in comparable locations in cKO brains conversely reveals alarge number of DiD-labeled axons (green arrowheads) that ectopically label cells in the dLG, adjacent to DiI-labeled cells (red arrowheads). Only some DiD-labeled cells are present in the VPN in cKO brains.
Fig. S4. Lhx2 is expressed in cortical basal progenitors and upper-layer neurons. (A) Immunostaining on P7 sagittal sections reveals that Lhx2 expressionoverlaps with Satb2 expression in upper cortical layers (layers 2–4), but Lhx2 expression is absent in Ctip2-expressing layer 5 neurons. Numbers indicate corticallayers. (B) Double labeling of E15 coronal sections using Lhx2 and Tbr2 antibodies demonstrates that Lhx2 is coexpressed in Tbr2-positive basal progenitors inthe SVZ (merged image). Arrowheads point to representative double-positive basal progenitors.
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Fig. S5. Lhx2 is transiently expressed in early postmitotic layer 5 neurons. Immunolabeling on E15 (A) and E14 (B) coronal sections reveals Lhx2 expression inthe VZ, IZ, and CP, whereas a Ctip2 antibody stains newly born layer 5 neurons in the CP. Lhx2/Ctip2 double labeling (merged image) reveals that Lhx2 iscoexpressed in a subpopulation of Ctip2-positive layer 5 neurons in the CP at both stages. Arrowheads point to representative double-positive layer 5 neurons.
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Fig. S6. Lhx2 is transiently expressed in early postmitotic layer 6 neurons. Immunolabeling on E13 (A) and E12 (B) coronal sections reveals Lhx2 expression inthe VZ, IZ, PP, and CP, whereas a Tbr1 antibody stains newly born layer 6 neurons in the PP/CP. Lhx2/Tbr1 double labeling (merged image) reveals that Lhx2is coexpressed in a subpopulation of Tbr1-positive layer 6 neurons in the PP/CP at both stages. Arrowheads point to representative double-positive layer6 neurons.
Fig. S7. Summary of cortical Lhx2 expression in WT and after conditional Lhx2 deletion from cortical neurons. In WT, Lhx2 is permanently expressed in corticalprogenitors in the VZ/SVZ (red cells) and in layer 2–4 neurons (blue cells) embryonically. Postnatally, permanent Lhx2 expression persists in neurons in layers 2–4(blue cells). In addition, Lhx2 is transiently expressed in subpopulations of layer 5 (green cells) and layer 6 (light green cells) neurons after they becomepostmitotic and start to migrate to form the CP. In cKO, Lhx2 expression remains unchanged in cortical progenitors (red cells) throughout development but isspecifically deleted from all postmitotic neurons. E, ependyma. Numbers indicate cortical layers.
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Fig. S8. MeCP2 and HDAC1 staining appears normal in subcortical brain regions. Immunolabeling forMeCP2 and HDAC1 on P7 sagittal sections at the level ofthe thalamus (MeCP2) and the cerebellum (HDAC1) reveals comparable staining intensities in WT and cKO brains.
Fig. S9. Downstream regulators are already affected at P0, before sensory areas emerge. ISH on sagittal P0 sections reveals the expression of marker genes.Dotted lines identify the exemplary CP regions that are shown in the high-magnification images. (A) In WT sections, the ISH and immunostainings for Bhlhb5,Lmo4, and CoupTF1 in the CP start to reveal differences in the expression levels among cortical areas, although the expression borders are still very fuzzy.Bhlhb5 and Lmo4 expression appears down-regulated in cKO sections, compared with WT sections. Conversely, CoupTF1 expression in the caudal cortexappears stronger in cKO sections, with its high-expression domain extending much more rostrally than in WT sections. (B) Immunostaining of the markersMeCP2, HDAC1, and 5Mc on WT sections at P0 shows staining in many cortical cells but does not reveal significant staining differences among cortical areas.Some differential expression is evident among cortical layers. Staining for MeCP2 appears stronger in layers 2–3 and 6 than in layers 4–5, whereas HDAC1 and5Mc immunolabelings appear stronger in layers 2–3 than in deeper cortical layers. In cKO sections, the labelings for MeCP2 and HDAC1 appear less intense,especially in upper cortical layers. 5Mc staining in cKO sections is largely comparable to that in WT sections in the CP, except that lower staining is evident incortical layers 2–3.
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Fig. S10. Sequential model for area patterning in progenitors and postmitotic cortical neurons. According to the current model, area identity is specified incortical progenitors by the cooperated activity of patterning TFs such as Emx2, Pax6, CoupTF1, and Sp8 (1–3). According to the radial unit hypothesis (4), areapatterning is linearly transferred to daughter neurons that form the CP simultaneously in a one-step process. The intermediate genetic regulatory elements,including the TFs Lmo4 and Bhlhb5 (1), act in neurons postmitotically and participate in the acquisition of select area features in specific cell types, layers, orareas in the CP without changing overall area patterning determined at the level of progenitors. Our study suggests that the establishment of area patterningin neurons in the CP requires additional mechanisms that operate in postmitotic neurons. In cKO cortices that specifically lack Lhx2 function in postmitoticcortical neurons, area identity in some regions of the CP (e.g., enlarged V1, contracted S1) is different from how it was specified initially in VZ progenitors,demonstrating that the specification of area identity in VZ progenitors (1–3) alone is insufficient to specify the full array of functional area properties in the CP.We suggest a revised and more comprehensive model of area patterning that requires two steps. After initial specification of area identity in progenitors bypatterning TFs, we propose a crucial requirement for sustained activity of key alignment TFs, including Lhx2, which is required postmitotically to regulate areaidentity in the cortex to transfer linearly and coordinate area properties in neurons by controlling the entire array of area-specific features postmitotically. Wepropose a novel molecular hierarchy that regulates global sensory area features in cortical postmitotic neurons. In this model, Lhx2 acts on top of a regulatorynetwork that influences the activity of downstream intermediate regulatory elements including transcription factors and epigenetic regulators that are re-sponsible for cooperatively controlling gene expression in the CP required to establish sensory area properties in adult sensory areas.
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