Supporting Information

Bedogni et al. 10.1073/pnas.1002285107 SI Methods vortexing, aqueous solution containing total RNA was separated Animals and Tissue-Collection Procedures. Tbr1 mutant mice were by high-speed centrifugation at 4 °C for 20 min. RNA was pre- maintained as heterozygotes on CD1 background. To generate cipitated by adding an equal volume of isopropanol, and it was Tbr1 homozygous null animals, heterozygous males were crossed stored at −20 °C overnight and then, pelleted by centrifugation for with heterozygous females. The day of the vaginal plug was con- 20 min at maximum speed at 4 °C, rinsed with 70% ethanol, and sidered embryonic day (E) 0.5. Pregnant dams were deeply anes- resuspended in an appropriate volume of nuclease-free water. thetized with Avertin (3-3-tribromoethanol; Sigma) and killed by RNA samples were quality controlled with an Agilent 2100 Bio- cervical dislocation, after which embryos were harvested. For im- analyzer microfluidics-based system. Only RNA samples that passed munological analysis or in situ hybridization, embryos were rapidly QC were further processed. The University of Washington National removed and perfused with 4% paraformaldehyde in 1× PBS (pH Institution on Environmental Health Sciences Center for Ecoge- 7.4). Embryonic brains were postfixed by immersion in cold buff- netics and Environmental Health Functional Genomics Laboratory ered (4 °C) 4% paraformaldehyde for 4–16 h. Brains were cry- followed the manufacturer’s protocols for the determination of gene oprotected in 30% sucrose in 0.1 M sodium phosphate (pH 7.0), expression using the GeneChip Mouse Genome 430 2.0 Array plat- frozen in optimum cutting temperature compound (Sakura Fine- form by Affymetrix for E14.5 samples and the Mouse Gene 1.0 ST tek), cryosectioned at 12 μm, and mounted on Superfrost Plus slides Array (Affymetrix) for P0 samples. These methods include the syn- (Thermo Fisher Scientific). Slides were stored at −80 °C until thesis of first- and second-strand cDNAs, the purification of double- needed. All experimental procedures were approved by the Uni- stranded cDNA, the synthesis of cRNA by in vitro transcription, the versity of Washington and Seattle Children’s Research Institute recovery and quantitation of biotin-labeled cRNA, the fragmenta- Institutional Animal Care and Use Committees. tion of this cRNA and subsequent hybridization to the microarray slide, the posthybridization washings, and the detection of the hy- Probes for in Situ Hybridization. Riboprobes were generated from bridized cRNAs using streptavidin-coupled fluorescent dye. Hybrid- cDNA obtained from the following sources: Auts2 from Open Bi- ized Affymetrix arrays were scanned with an Affymetrix GeneChip osystems, Bcl6 from Open Biosystems, Rorb from the Rubenstein 3000 scanner. Image generation and feature extraction were per- lab (University of California, San Francisco, CA), Etv5 from Eliz- formed using Affymetrix GCOS Software. abeth Grove (University of Chicago, Chicago, IL), Cdh9 from Open Raw microarray data were processed and analyzed with Bio- Biosystems, Crym from Paola Arlotta (Harvard Medical School, conductor and normalized using the robust multichip average Boston, MA), Lmo4 from Tao Sun (Cornell Medical School, New (RMA) method from the Bioconductor Affy package. Using the York, NY), Epha6 from Maria Donoghue (Georgetown Univer- normalized data, genes with significant evidence for differential sity, Washington, DC), Tshz2 from Xavier Caubit (University of expression were identified with the limma package in Biocon- Marseilles, Marseilles, France), Odz3 from Dennis O’Leary (Uni- ductor. P values were calculated with a modified t test in con- versity of California, San Diego, CA), Pvrl3 from Open Biosystems, junction with an empirical Bayes method to moderate the SEs of Pcdh8 from Open Biosystems, Er81 from Silvia Arber (University of the estimated log-fold changes. P values were adjusted for mul- Basel, Basel, Switzerland), Sox5 from Maike Sander (University of tiplicity with the Bioconductor package q value, which allows for California, Irvine, CA), Crim1 from Jeffrey Macklis (Harvard Med- selecting statistically significant genes while controlling the es- ical School, Boston, MA), Fezf2 from the Rubenstein lab, Mef2c timated false-discovery rate. from Carol Schuurmans (University of Calgary, Calgary, Canada), and Ctgf from Open Biosystems. Gene-Set Analysis of Microarray Data. We used gene-set analysis (GSA) to investigate categories of genes where the constituent Primary Antibodies for Immunohistochemistry. Primary antibodies genes show coordinated changes in expression over the experi- were used as follows from these sources: Tbr1 (rabbit, 1:1,000) from mental conditions. GSA is a recently established category/pathway theHevnerlab,Sox5(rabbit,1:500)fromSantaCruzBiotechnology, analysis method, and GSA software is freely available as R code. Tle4 (rabbit, 1:1,000) from Stefano Stifani, Er81 (rabbit, 1:2,500) GSA considers all genes in the experiment and allows for the from Silvia Arber, Brn2 (rabbit, 1:500) from Santa Cruz Bio- identification of gene sets/pathways with genes that show modest technology, activated caspase-3 (rabbit, 1:500) from Santa Cruz but concordant changes in gene expression. We used five databases Biotechnology, FOG2 (rabbit, 1:500) from Santa Cruz Bio- of gene sets for GSA, three from the Gene Ontology Database technology, Auts2 (rabbit, 1:1,000) from Sigma, GFP (chicken, (Biological Process, Molecular Function, and Cellular Compo- 1:500)fromAbcam,BrdU(mouse,1:200)fromMillipore,BrdU(rat, nent) and two from the Molecular Signature Database. In addition, 1:400) from Accurate Chemical and Scientific, Ctip2 (rat, 1:1,000) we used custom gene sets for regional and laminar identity (Tables fromAbcam,NeuroD(goat,1:400)fromSantaCruzBiotechnology, S1– S4). GSA was applied to custom gene sets to test for statistical and Bhlhb5 (goat, 1:500) from Santa Cruz Biotechnology. changes in groups of genes representing regional or laminar identity in Tbr1 mutant compared with control cortex. RNA Isolation and Microarrays. E14.5 neocortices were rapidly col- lected under a dissecting microscope, frozen on dry ice, and stored Ex Utero Electroporation. Embryos were harvested from timed- at −80 °C for later RNA isolation. Postnatal day (P) 0.5 brains were pregnant females on E13.5 and immersed in artificial cerebrospinal rapidly dissected and cut into coronal slabs ∼1-mm thick; regions fluid(aCSF).PlasmidDNAdilutedtoafinalconcentrationof1μg/μL corresponding to frontal, parietal, and occipital cortex were mi- (p-iresGFP for control injections or p-Tbr1iresGFP) was injected crodissected, frozen on dry ice, and stored at −80 °C. Total RNA into the lateral ventricle using a glass micropipette. Six 50-ms pulses purification was performed as described (1). Briefly, tissues were (35 mV) were applied at an interval of 950 ms using platinum paddle dissolved by low-power sonication in a solution consisting of 4 M electrodes. Electroporation was performed using a BTX Electro guanidinium thiocyanate and 0.1 M 2-mercaptoethanol. After Square Electroporator ECM830. After electroporation, brains were sonication, sodium acetate (pH 4), phenol (pH 4), and a 24:1 mix of dissected and transferred to ice-cold aCSF. The forebrain was em- chloroform and isoamylic acid were added on ice; after vigorous bedded in 4% low-melting temperature agarose(Seaplaque; Lonza)

Bedogni et al. www.pnas.org/cgi/content/short/1002285107 1of11 dissolvedinaCSF.Agaroseblocksweresectionedat400μmon not been defined. PCR primers were designed to amplify the a vibratome (Leica), and individual slices were transferred onto candidate Tbr1 binding sites as well as adjacent sequences lacking culture membrane inserts (Millicell; Millipore). Slices were cultured T-box transcription factors (TF) binding sites (as negative con- × overnight in Neurobasal Media (Invitrogen) supplemented with 1 trols) in the putative Auts2 regulatory region (Fig. 6I). × × B27, 1 N2, 1 L-glutamine, 100 U/mL penicillin-streptomycin Neocortex was dissected from E14.5 embryos as in microarray (Invitrogen), and 5% FBS (Mediatech). experiments. Chromatin cross-linking, isolation, sonication, and antibody binding were done as described (4). DNA was purified ChIP. Auts2 gene sequences were scanned for evolutionary conserved regions (ECRs) from aligned vertebrate genomes using using EZ-ChIP (Millipore) and then, analyzed for candidate Tbr1 ECR Browser (2). Potential Tbr1 binding sites were identified binding sites and adjacent sequences in the Auts2 regulatory re- using TRANSFAC Professional v10.2 library of position-weight gion using quantitative PCR (qPCR) assays. ChIP with antibodies matrices (3). The Tbx5 matrix was used, because it is represen- against acetylated histones (identifying transcriptionally active tative of conserved T-box binding sites and the Tbr1 matrix has chromatin) were used as a positive control.

1. Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid 3. Wingender E, Dietze P, Karas H, Knüppel R (1996) TRANSFAC: A database on guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159. transcription factors and their DNA binding sites. Nucleic Acids Res 24:238–241. 2. Ovcharenko I, Nobrega MA, Loots GG, Stubbs L (2004) ECR Browser: A tool for visualizing 4. Beima KM, et al. (2006) T-bet binding to newly identiﬁed target gene promoters is cell and accessing data from comparisons of multiple vertebrate genomes. Nucleic Acids Res type-independent but results in variable context-dependent functional effects. J Biol 32:W280–W286. Chem 281:11992–12000.

Fig. S1. Tbr1 expression in developing neocortex compared with Bhlhb5. Tbr1 (A, F, L, and Q;redinC–E, H–K, N–P,andS–U) was highly expressed in rostral cortex and early-born neurons [Cajal-Retzius (C-R), subplate (SP), and layer 6]. Bhlhb5 was expressed in a complementary pattern that was highest in caudal and late-born (layers 2–5) neurons (B, G, M, and R and green in C–E, H–K, N–P, and S–U). Parasagittal (rostral left; A–C, F–H, L–N, and Q–S) and coronal sections (D, E, I, K, O, P, T, and U) are shown. Ages are as indicated. (Scale bar: 400 μm.)

Bedogni et al. www.pnas.org/cgi/content/short/1002285107 2of11 Fig. S2. Some FGF signaling components are up-regulated in Tbr1 mutant cortex. (A–L) In situ hybridization (ISH) in coronal sections on E12.5 showed up- regulation of Fgf15 and more robustly, Fgf17 in medial rostral cortex of Tbr1 mutants. Among FGF signaling reporters, Spry1 was markedly increased in medial and dorsal rostral cortex of the mutant (F), whereas Spry2, Etv1, and Etv5 were affected little or not at all. (M–T) Sagittal sections through dorsal cortex on E14.5 showed increased gene expression of Spry1 in the mutant ventricular zone (VZ) (N), with little discernible effect on Spry2, Etv1, and Etv5. On microarray comparing Tbr1 null to control E14.5 cortex, Fgf15, Fgf17, and Spry1 were markedly up-regulated, but Spry2, Etv1, Etv5 and Fgf8 were not. Genotypes are as indicated. [Scale bars (A–L): 200 μm; (M–T): 400 μm.]

Fig. S3. Progenitor TF gradients are unchanged in Tbr1 mutant cortex. (A–D) ISH in E12.5 sagittal sections showed no change in the Pax6 rostrocaudal gradient and no signiﬁcant alteration of Tbr2 expression. (E–J) Likewise, E14.5 sagittal sections showed no change in the rostrocaudal Pax6 gradient or the caudorostral CoupTF1 gradient. Tbr2 expression was perturbed only in the region of the malformed olfactory bulb. By microarray, expression of these genes was not signiﬁcantly different in Tbr1 null versus control E14.5 cortex. Genotypes are as indicated. (Scale bar: 400 μm.)

Bedogni et al. www.pnas.org/cgi/content/short/1002285107 3of11 Fig. S4. Apoptosis was increased in frontal cortex of Tbr1 mutant mice. (A–F) Parasagittal sections immunolabeled for active caspase-3 (red) to detect cell death and counterstained with the nuclear marker DAPI (blue). a′–f′ and a″ –f″ are higher magniﬁcation views of the boxed regions in A–F in frontal areas (a′–f′) and parietal areas (a″–f″), showing the initiation of cell death at E16.5 (A and B) and progressive increase in cell death at E18.5 (C and D) and P0.5 (E and F)inTbr1 mutant mice (B, D, and F) compared with controls (A, C,andE). Frontal areas (a′–f′) showed a greater increase in active caspase-3 positive cells than did parietal areas (a″–f″)inTbr1 mutant cortices. [Scale bars (A–F): 200 μm; (a′–f′ and a″–f″): 25 μm.]

Fig. S5. SP and CP markers were decreased in E14.5 Tbr1 mutant cortex. (A–F) Expression of SP marker Mef2c (A and B) and cortical plate (CP) markers Sox5 (C and D) and Ctip2 (E and F) were decreased in E14.5 Tbr1 knockout (KO; B, D, and F) compared with control (A, C, and E) neocortex. Mef2c expression was detected by ISH, and Sox5 and Ctip2 expression was detected by immunohistochemistry. Microarray analysis of these genes (G)conﬁrmed the signiﬁcantly decreased expression of these genes in Tbr1 mutant cortices. [Scale bars (A–F): 400 μm.]

Bedogni et al. www.pnas.org/cgi/content/short/1002285107 4of11 Fig. S6. Layer 5 identity increased and layer 6 identity decreased in Tbr1 mutant cortex. (A–L) The molecular fate index of early-born cells (labeled with BrdU on E12.5) was determined by counting the percentage of BrdU+ cells that expressed each marker of layers 5 and 6 after survival to P0.5. The Sox5 fate index (A–C) was not signiﬁcantly changed, but Sox5 is expressed in both layer 6 (Sox5 high) and layer 5 (Sox5) and counts did not distinguish levels of expression. The Tle4 (layer 6) fate index was reduced in Tbr1 mutant parietal and occipital cortex (D–F). Conversely, the fate index of layer 5 markers Er81 (G–I) and Ctip2 (J–L) was increased in Tbr1 mutant cortex. Tbr1 genotypes are as indicated. (M and N) Axon tracing with DiI (red/pink) was injected in the frontal cortex (injection sites not shown) to label afferent and efferent projections in P0.5 control (M) and Tbr1 mutant (N) brains. Compared with controls, Tbr1 mutants had reduced connections with the thalamus (Th), characteristic of layer 6, and increased projections to the cerebral peduncle (CeP), characteristic of layer 5. Ectopic projections into the hypothalamus (Hy) were also observed in Tbr1 mutants. (O) Laminar fate is regulated by a network of TF interactions. The dashed red arrow from Tbr1 to Sox5 suggests a direct or indirect mechanism of transcriptional activation. Adapted from Leone et al. (1). *P < 0.05. [Scale bars (A–K): 50 μm; (M and N): 500 μm.]

1. Leone DP, Srinivasan K, Chen B, Alcamo E, McConnell SK (2008) The determination of projection neuron identity in the developing cerebral cortex. Curr Opin Neurobiol 18:28–35.

Bedogni et al. www.pnas.org/cgi/content/short/1002285107 5of11 Fig. S7. Tbr1 overexpression by plasmid electroporation ex utero. (A–L′) Electroporation of Tbr1 cDNA expression plasmid (Tbr1–ires–GFP), but not GFP only (ires–GFP), drove ectopic expression of Tbr1 (red) in 92.6 ± 1.8% of GFP+ (green) cells in control (A–C′ and G–I′) and 91.6 ± 2.6% of total GFP+ cells in Tbr1 mutant (D–F′ and J–L′) VZ. The VZ from C, F, I, and L is shown at higher magniﬁcation in C′, F′, I′, and L′. [Scale bars (A–F): 50 μm; (C′ and F′): 30 μm; (G–L): 100 μm; (I′ and L′): 25 μm.]

Bedogni et al. www.pnas.org/cgi/content/short/1002285107 6of11 Table S1. Regional gene-expression markers in developing mouse cerebral neocortex Region-zone Gene Laminar speciﬁcity References

Rostral-CP/intermediate zone Auts2* CP 1 (IZ)/SP (n =9) Bcl6 IZ, CP 2 and 3 Fat3* CP 4 Nrip1* CP 4 Ppp1r1b (DARPP-32) CP 2 and 5 Ptpro* CP 4 Rorb CP 2 and 6 Spry2 CP 4 and 7 Tbr1 CP 8 Rostral-subventricular zone Cdh10* SVZ > SP 4 (SVZ) (n =3) Cdh8* SVZ > CP 2 and 4 Tbr2 (Eomes) SVZ 9 Rostral-VZ (n =8) Dct VZ 4 Elk3* VZ 4 Etv1 (Er81) VZ 4 and 7 Etv5 (ERM) VZ 7 Mest VZ 4 and 7 Pax6 VZ 10 Rlbp1 VZ 4 and 7 Sema5a VZ 4 and 11 Caudal-CP/IZ/SP (n =7) Bhlhe22 (Bhlhb5) CP > IZ/SVZ 7 and 12 Cdh11 (OB-cad)* CP 4 and 7 Crym* CP 4 Nhlh1 (Nscl)* IZ 4 Odz1 SP > CP 13 Odz2 CP, SP 7 and 13 Odz3 (Ten_m3) CP, SP 7 and 14 Caudal-SVZ (n =1) Tcf4 (ME2) SVZ/IZ > CP 2 and 13 Caudal-VZ (n =4) Bmp7 VZ 4 and 15 Emx2 VZ 10 Fgfr3 VZ 4 and 16 Nr2f1 (COUP-TF1) VZ 2 and 17

E13.5–E15.5 neocortex. Genepaint (4) data from E14.5; Allen brain map (2) data from E13.5 or E15.5. *Newly identiﬁed rostral or caudal markers in this age range.

1. Bedogni F, et al. (2010) Autism susceptibility candidate 2 (Auts2) encodes a nuclear protein expressed in developing brain regions implicated in autism neuropathology. Gene Expr Patterns 10:9–15. 2. Lein ES, et al. (2007) Genome-wide atlas of gene expression in the adult mouse brain. Nature 445:168–176. 3. Funatsu N, Inoue T, Nakamura S (2004) Gene expression analysis of the late embryonic mouse cerebral cortex using DNA microarray: Identification of several region- and layer-specific genes. Cereb Cortex 14:1031–1044. 4. Alvarez-Bolado G, Eichele G (2006) Analysing the developing brain transcriptome with the GenePaint platform. J Physiol 575:347–352. 5. Mühlfriedel S, Kirsch F, Gruss P, Chowdhury K, Stoykova A (2007) Novel genes differentially expressed in cortical regions during late neurogenesis. Eur J Neurosci 26:33–50. 6. Zhou C, Tsai SY, Tsai MJ (2001) COUP-TFI: An intrinsic factor for early regionalization of the neocortex. Genes Dev 15:2054–2059. 7. Sansom SN, et al. (2005) Genomic characterisation of a Fgf-regulated gradient-based neocortical protomap. Development 132:3947–3961. 8. Bulfone A, et al. (1995) T-brain-1: A homolog of Brachyury whose expression defines molecularly distinct domains within the cerebral cortex. Neuron 15:63–78. 9. Bulfone A, et al. (1999) Expression pattern of the Tbr2 (Eomesodermin) gene during mouse and chick brain development. Mech Dev 84:133–138. 10. Bishop KM, Goudreau G, O’Leary DD (2000) Regulation of area identity in the mammalian neocortex by Emx2 and Pax6. Science 288:344–349. 11. Jones L, López-Bendito G, Gruss P, Stoykova A, Molnár Z (2002) Pax6 is required for the normal development of the forebrain axonal connections. Development 129:5041–5052. 12. Joshi PS, et al. (2008) Bhlhb5 regulates the postmitotic acquisition of area identities in layers II-V of the developing neocortex. Neuron 60:258–272. 13. Li H, Bishop KM, O’Leary DD (2006) Potential target genes of EMX2 include Odz/Ten-M and other gene families with implications for cortical patterning. Mol Cell Neurosci 33:136–149. 14. Zhou XH, et al. (2003) The murine Ten-m/Odz genes show distinct but overlapping expression patterns during development and in adult brain. Gene Expr Patterns 3:397–405. 15. Furuta Y, Piston DW, Hogan BL (1997) Bone morphogenetic proteins (BMPs) as regulators of dorsal forebrain development. Development 124:2203–2212. 16. Muzio L, et al. (2002) Emx2 and Pax6 control regionalization of the pre-neuronogenic cortical primordium. Cereb Cortex 12:129–139. 17. Liu Q, Dwyer ND, O’Leary DD (2000) Differential expression of COUP-TFI, CHL1, and two novel genes in developing neocortex identified by differential display PCR. J Neurosci 20: 7682–7690.

Bedogni et al. www.pnas.org/cgi/content/short/1002285107 7of11 Table S2. Regional gene-expression markers in developing mouse cerebral neocortex Region Gene Layers References

Rostral (n = 28) Auts2* L2–61 Bcl6 L2–3high 2–4 Bhlhe40 (Bhlhb2)* L2–3high 2 Btbd11* L2–4 high 2 and 5 Cdh10 All [callosal projection 2and6 neuron (CPN)] Cdh4 (R-cad) All 7 Cdh9* L6 2 Dusp6 Layers? 4 Etv5 (ERM) L4–62and8 Fam84a CP > SVZ 5 Fat3* L2-5 2 Klhl29 (Kbtbd9) Layers? 4 Luzp2 Layers? 4 Mc4r* All layers 2 Mkx (Irxl1)* L2–3hi 2 Pappa2* Upper L 2 Pcdh20 L2–4hi 2and9 Ppp1r1b (DARPP-32) L6, SP 2 and 5 Ptprk All layers 2 and 3 Ptpro Layers? 4 Rorb L4 4, 10, and 11 Scrt2 SVZ 15 Sfrp2 L6 2 and 12 Tbr1 L2–6 4, 10, and 12 Tgfbr1* L2–52–4 Tox* L5 > other 2 and 5 Wscd1 L6, SP 5 Zfp385b* Layers? Current project Caudal (n = 30) Bhlhe22 (Bhlhb5) Hi L5, Lo 2–4 2 and 13 Cadm1* L5–62and4 Chodl* Layers? 5 Cntn3* L5 2 Cntn6 (NB-3) L5 subcerebral projection 2, 6, and 14 neuron (SCPN) Crym* L5 > L6 2 and 4 Dkk3 L2–5 CPN 2, 4, and 6 Epha6 L2–5 2 and 15 Flrt3 L2–34and5 Fn1* L2–42 Lhx2 L2/3, 5, 6 2 and 16 Met (AI838057)* All layers 2 and 4 Neﬂ* Layers? 5 Nefm* L2–35 Neurod1 L2–32and4 Ngfr (p75NTR) L6/SP 2, 13, and 17 Nr2f1 (COUP-TF1) L4 > all L 2 and 18 Nr4a2 (Nurr1) All (intra ctx–ctx) 5 and 19 Nrp1 L6 > L2–42and4 Ntng2* Upper L 2 Nts* L5 2 Odz2 All layers 4 and 20 Odz3 (Ten-m3) L5 > other L 4, 20, and 21 Odz4 All layers 4 and 20 Pcdh8 L2–3 > 59 Ptprz1* L2–32 Pvrl3 (nectin-3)* L2–3, CPN 2 and 22 Tshz2* Layers? 23 Tshz3* Layers? 4 and 23 Vat1l* Layers? 5

E18.5–P4 E15 CP/SP. Allen brain map (2) data from E18.5 or P4 cortex. *Newly identiﬁed rostral or caudal markers in this age range.

Bedogni et al. www.pnas.org/cgi/content/short/1002285107 8of11 1. Bedogni F, et al. (2010) Autism susceptibility candidate 2 (Auts2) encodes a nuclear protein expressed in developing brain regions implicated in autism neuropathology. Gene Expr Patterns 10:9–15. 2. Lein ES, et al. (2007) Genome-wide atlas of gene expression in the adult mouse brain. Nature 445:168–176. 3. Funatsu N, Inoue T, Nakamura S (2004) Gene expression analysis of the late embryonic mouse cerebral cortex using DNA microarray: Identification of several region- and layer-specific genes. Cereb Cortex 14:1031–1044. 4. Leamey CA, et al. (2008) Differential gene expression between sensory neocortical areas: Potential roles for Ten_m3 and Bcl6 in patterning visual and somatosensory pathways. Cereb Cortex 18:53–66. 5. Mühlfriedel S, Kirsch F, Gruss P, Chowdhury K, Stoykova A (2007) Novel genes differentially expressed in cortical regions during late neurogenesis. Eur J Neurosci 26:33–50. 6. Arlotta P, et al. (2005) Neuronal subtype-specific genes that control corticospinal motor neuron development in vivo. Neuron 45:207–221. 7. Faedo A, et al. (2008) COUP-TFI coordinates cortical patterning, neurogenesis, and laminar fate and modulates MAPK/ERK, AKT, and beta-catenin signaling. Cereb Cortex 18: 2117–2131. 8. Rash BG, Grove EA (2006) Area and layer patterning in the developing cerebral cortex. Curr Opin Neurobiol 16:25–34. 9. Kim SY, Chung HS, Sun W, Kim H (2007) Spatiotemporal expression pattern of non-clustered protocadherin family members in the developing rat brain. Neuroscience 147:996–1021. 10. Miyashita-Lin EM, Hevner R, Wassarman KM, Martinez S, Rubenstein JL (1999) Early neocortical regionalization in the absence of thalamic innervation. Science 285:906–909. 11. Nakagawa Y, O’Leary DD (2003) Dynamic patterned expression of orphan nuclear receptor genes RORalpha and RORbeta in developing mouse forebrain. Dev Neurosci 25:234–244. 12. Fukuchi-Shimogori T, Grove EA (2001) Neocortex patterning by the secreted signaling molecule FGF8. Science 294:1071–1074. 13. Joshi PS, et al. (2008) Bhlhb5 regulates the postmitotic acquisition of area identities in layers II-V of the developing neocortex. Neuron 60:258–272. 14. Chen B, Schaevitz LR, McConnell SK (2005) Fezl regulates the differentiation and axon targeting of layer 5 subcortical projection neurons in cerebral cortex. Proc Natl Acad Sci USA 102:17184–17189. 15. Yun ME, Johnson RR, Antic A, Donoghue MJ (2003) EphA family gene expression in the developing mouse neocortex: Regional patterns reveal intrinsic programs and extrinsic influence. J Comp Neurol 456:203–216. 16. Nakagawa Y, Johnson JE, O’Leary DD (1999) Graded and areal expression patterns of regulatory genes and cadherins in embryonic neocortex independent of thalamocortical input. J Neurosci 19:10877–10885. 17. McQuillen PS, DeFreitas MF, Zada G, Shatz CJ (2002) A novel role for p75NTR in subplate growth cone complexity and visual thalamocortical innervation. J Neurosci 22:3580–3593. 18. Liu Q, Dwyer ND, O’Leary DD (2000) Differential expression of COUP-TFI, CHL1, and two novel genes in developing neocortex identified by differential display PCR. J Neurosci 20: 7682–7690. 19. Arimatsu Y, Ishida M, Kaneko T, Ichinose S, Omori A (2003) Organization and development of corticocortical associative neurons expressing the orphan nuclear receptor Nurr1. J Comp Neurol 466:180–196. 20. Li H, Bishop KM, O’Leary DD (2006) Potential target genes of EMX2 include Odz/Ten-M and other gene families with implications for cortical patterning. Mol Cell Neurosci 33:136–149. 21. Zhou XH, et al. (2003) The murine Ten-m/Odz genes show distinct but overlapping expression patterns during development and in adult brain. Gene Expr Patterns 3:397–405. 22. Molyneaux BJ, et al. (2009) Novel subtype-specific genes identify distinct subpopulations of callosal projection neurons. J Neurosci 29:12343–12354. 23. Caubit X, Tiveron MC, Cremer H, Fasano L (2005) Expression patterns of the three Teashirt-related genes define specific boundaries in the developing and postnatal mouse forebrain. J Comp Neurol 486:76–88.

Table S3. Markers of laminar or neuron subtype identity in developing mouse neocortex Neuron type Gene Regions References

Cajal-Retzius (n =3) Calb2 (Calretinin) All 1 and 2 Reln (Reelin) All 1 and 2 Trp73 (p73) All 2 and 3 Subplate (n =3) Kitl* All 4 Mef2c* All 4 Odz1 Caudal 5 Cortical plate (n =5) Bcl11b (Ctip2) All 6 and 7 Cdh13* All 4 and 6 Cnr1 All 6 and 8 Sox5 All 6 and 9 Zeb2 (SIP1) All 6 and 10

E13.5–E15.5 neocortex. Genepaint (6) data from E14.5; Allen brain map (4) data from E13.5 or E15.5. *Genes newly identiﬁed as speciﬁc neuron subtype markers in this age range.

1. Alcántara S, et al. (1998) Regional and cellular patterns of reelin mRNA expression in the forebrain of the developing and adult mouse. J Neurosci 18:7779–7799. 2. Yamazaki H, Sekiguchi M, Takamatsu M, Tanabe Y, Nakanishi S (2004) Distinct ontogenic and regional expressions of newly identified Cajal-Retzius cell-specific genes during neocorticogenesis. Proc Natl Acad Sci USA 101:14509–14514. 3. Meyer G, Perez-Garcia CG, Abraham H, Caput D (2002) Expression of p73 and Reelin in the developing human cortex. J Neurosci 22:4973–4986. 4. Lein ES, et al. (2007) Genome-wide atlas of gene expression in the adult mouse brain. Nature 445:168–176. 5. Li H, Bishop KM, O’Leary DD (2006) Potential target genes of EMX2 include Odz/Ten-M and other gene families with implications for cortical patterning. Mol Cell Neurosci 33:136–149. 6. Alvarez-Bolado G, Eichele G (2006) Analysing the developing brain transcriptome with the GenePaint platform. J Physiol 575:347–352. 7. Arlotta P, et al. (2005) Neuronal subtype-specific genes that control corticospinal motor neuron development in vivo. Neuron 45:207–221. 8. Berghuis P, et al. (2007) Hardwiring the brain: Endocannabinoids shape neuronal connectivity. Science 316:1212–1216. 9. Lai T, et al. (2008) SOX5 controls the sequential generation of distinct corticofugal neuron subtypes. Neuron 57:232–247. 10. Seuntjens E, et al. (2009) Sip1 regulates sequential fate decisions by feedback signaling from postmitotic neurons to progenitors. Nat Neurosci 12:1373–1380.

Bedogni et al. www.pnas.org/cgi/content/short/1002285107 9of11 Table S4. Markers of laminar or neuron subtype identity in developing mouse neocortex Layer Gene Regions References

L2–3(n =11) Flrt3* Caudal 1 and 2 Limch1 All 3 Mdga1 Caudal 4 and 5 Mef2c All 4 and 6 Nefm* Caudal 1 Pcdh8 Caudal 7 Ptprz1* Caudal 4 Pvrl3 Caudomedial 3 and 4 Rgs8* All Current project Sorl1 All 8 and 9 Tle1 All 10 L2–4(n =10) Cux1 All 4 and 11 Cux2 All 4, 11, and 12 Dtx4 All 4 and 6 Inhba All 4 and 3 Nrgn* All 3 Pcdh11x All 7 Pcdh20 Rostral 4 and 7 Pou3f2 (Brn-2) All 4, 9, and 10 Pou3f3 (Brn-1) All 9 and 10 Unc5d (Svet1) All 6, 13, and 14 L4 (n =2) Rorb Rostral 2, 15, and 16 Unc5c* All 4 CPN (n =6) Dkk3* Caudomedial 4, 17, and 18 Inhba All 4 and 3 Limch1 All 3 Ptn All 17 and 18 Pvrl3* Caudomedial 3 and 4 Satb2 All 4, 13, and 18 L5 (n =8) Bhlhb5 Caudomedial 4, 19, and 20 Cdh8 Rostral + caudal 21 and 22 Cntn3* Caudal 4 Etv1 (Er81) All 23 and 24 Foxp1 All 4 and 25 Pcdh19 All 4 and 7 Pcp4 All 17 Tox Rostromedial 1 and 4 L5 SCPN (n =4) Ctip2 All 17 Cntn6 Caudal 17 Fezf2 All 26–28 Oma1 All 17 L5 corticospinal motor neuron Crim1 All 17 (CSMN; n =3) Crym Caudal 17 S100a10 All 17 L6 (n = 15) Cdh9* Rostral 4 Drd1a* All 4 Foxp2 All 4 and 25 Nfe2l3* All 4 Ngfr Caudal 4, 19, and 29 Npy All 4 and 30 Nr4a2 (Nurr1) Caudal 1, 31, and 32 Nr4a3 Caudal 1, 4, and 20 Ppp1r1b Rostral 1 and 4 Sox5 All 33 and 34 Tbr1 All 35 and 36 Tle4 All 4, 10, and 37 Wnt7b All 4 and 36 Wscd1* Rostral 1 Zfpm2 (FOG2) All 4 and 34 Subplate (n =2) Ctgf All 32 and 38 Nxph4* All 4 Cajal-Retzius (n =6) Calb2 All 39

Bedogni et al. www.pnas.org/cgi/content/short/1002285107 10 of 11 Table S4. Cont. Layer Gene Regions References

Cplx3* All Current project Lhx5 All 4, 20, and 40 Nhlh2* All 4 Reln All 39 Trp73 All 4 and 41

E18.5–P4 cortex. Allen brain map (4) data from E18.5 or P4 cortex. *Genes newly identiﬁed as laminar markers in this age range.

1. Mühlfriedel S, Kirsch F, Gruss P, Chowdhury K, Stoykova A (2007) Novel genes differentially expressed in cortical regions during late neurogenesis. Eur J Neurosci 26:33–50. 2. Leamey CA, et al. (2008) Differential gene expression between sensory neocortical areas: Potential roles for Ten_m3 and Bcl6 in patterning visual and somatosensory pathways. Cereb Cortex 18:53–66. 3. Molyneaux BJ, et al. (2009) Novel subtype-specific genes identify distinct subpopulations of callosal projection neurons. J Neurosci 29:12343–12354. 4. Lein ES, et al. (2007) Genome-wide atlas of gene expression in the adult mouse brain. Nature 445:168–176. 5. Takeuchi A, O’Leary DD (2006) Radial migration of superficial layer cortical neurons controlled by novel Ig cell adhesion molecule MDGA1. J Neurosci 26:4460–4464. 6. Zhong Y, et al. (2004) Identification of the genes that are expressed in the upper layers of the neocortex. Cereb Cortex 14:1144–1152. 7. Kim SY, Chung HS, Sun W, Kim H (2007) Spatiotemporal expression pattern of non-clustered protocadherin family members in the developing rat brain. Neuroscience 147:996–1021. 8. Hermans-Borgmeyer I, et al. (1998) Unique expression pattern of a novel mosaic receptor in the developing cerebral cortex. Mech Dev 70:65–76. 9. Sugitani Y, et al. (2002) Brn-1 and Brn-2 share crucial roles in the production and positioning of mouse neocortical neurons. Genes Dev 16:1760–1765. 10. McEvilly RJ, de Diaz MO, Schonemann MD, Hooshmand F, Rosenfeld MG (2002) Transcriptional regulation of cortical neuron migration by POU domain factors. Science 295:1528–1532. 11. Nieto M, et al. (2004) Expression of Cux-1 and Cux-2 in the subventricular zone and upper layers II-IV of the cerebral cortex. J Comp Neurol 479:168–180. 12. Zimmer C, Tiveron MC, Bodmer R, Cremer H (2004) Dynamics of Cux2 expression suggests that an early pool of SVZ precursors is fated to become upper cortical layer neurons. Cereb Cortex 14:1408–1420. 13. Tarabykin V, Stoykova A, Usman N, Gruss P (2001) Cortical upper layer neurons derive from the subventricular zone as indicated by Svet1 gene expression. Development 128: 1983–1993. 14. Britanova O, et al. (2008) Satb2 is a postmitotic determinant for upper-layer neuron specification in the neocortex. Neuron 57:378–392. 15. Miyashita-Lin EM, Hevner R, Wassarman KM, Martinez S, Rubenstein JL (1999) Early neocortical regionalization in the absence of thalamic innervation. Science 285:906–909. 16. Nakagawa Y, O’Leary DD (2003) Dynamic patterned expression of orphan nuclear receptor genes RORalpha and RORbeta in developing mouse forebrain. Dev Neurosci 25:234–244. 17. Arlotta P, et al. (2005) Neuronal subtype-specific genes that control corticospinal motor neuron development in vivo. Neuron 45:207–221. 18. Alcamo EA, et al. (2008) Satb2 regulates callosal projection neuron identity in the developing cerebral cortex. Neuron 57:364–377. 19. Joshi PS, et al. (2008) Bhlhb5 regulates the postmitotic acquisition of area identities in layers II-V of the developing neocortex. Neuron 60:258–272. 20. Gray PA, et al. (2004) Mouse brain organization revealed through direct genome-scale TF expression analysis. Science 306:2255–2257. 21. Fukuchi-Shimogori T, Grove EA (2001) Neocortex patterning by the secreted signaling molecule FGF8. Science 294:1071–1074. 22. Nakagawa Y, Johnson JE, O’Leary DD (1999) Graded and areal expression patterns of regulatory genes and cadherins in embryonic neocortex independent of thalamocortical input. J Neurosci 19:10877–10885. 23. Hevner RF, et al. (2003) Beyond laminar fate: Toward a molecular classification of cortical projection/pyramidal neurons. Dev Neurosci 25:139–151. 24. Yoneshima H, et al. (2006) Er81 is expressed in a subpopulation of layer 5 neurons in rodent and primate neocortices. Neuroscience 137:401–412. 25. Ferland RJ, Cherry TJ, Preware PO, Morrisey EE, Walsh CA (2003) Characterization of Foxp2 and Foxp1 mRNA and protein in the developing and mature brain. J Comp Neurol 460: 266–279. 26. Chen B, Schaevitz LR, McConnell SK (2005) Fezl regulates the differentiation and axon targeting of layer 5 subcortical projection neurons in cerebral cortex. Proc Natl Acad Sci USA 102:17184–17189. 27. Chen JG, Rasin MR, Kwan KY, Sestan N (2005) Zfp312 is required for subcortical axonal projections and dendritic morphology of deep-layer pyramidal neurons of the cerebral cortex. Proc Natl Acad Sci USA 102:17792–17797. 28. Molyneaux BJ, Arlotta P, Hirata T, Hibi M, Macklis JD (2005) Fezl is required for the birth and specification of corticospinal motor neurons. Neuron 47:817–831. 29. McQuillen PS, DeFreitas MF, Zada G, Shatz CJ (2002) A novel role for p75NTR in subplate growth cone complexity and visual thalamocortical innervation. J Neurosci 22:3580–3593. 30. Funatsu N, Inoue T, Nakamura S (2004) Gene expression analysis of the late embryonic mouse cerebral cortex using DNA microarray: Identification of several region- and layer-specific genes. Cereb Cortex 14:1031–1044. 31. Arimatsu Y, Ishida M, Kaneko T, Ichinose S, Omori A (2003) Organization and development of corticocortical associative neurons expressing the orphan nuclear receptor Nurr1. J Comp Neurol 466:180–196. 32. Watakabe A, et al. (2007) Comparative analysis of layer-specific genes in Mammalian neocortex. Cereb Cortex 17:1918–1933. 33. Lai T, et al. (2008) SOX5 controls the sequential generation of distinct corticofugal neuron subtypes. Neuron 57:232–247. 34. Kwan KY, et al. (2008) SOX5 postmitotically regulates migration, postmigratory differentiation, and projections of subplate and deep-layer neocortical neurons. Proc Natl Acad Sci USA 105:16021–16026. 35. Bulfone A, et al. (1995) T-brain-1: A homolog of Brachyury whose expression defines molecularly distinct domains within the cerebral cortex. Neuron 15:63–78. 36. Hevner RF, et al. (2001) Tbr1 regulates differentiation of the preplate and layer 6. Neuron 29:353–366. 37. Allen T, Lobe CG (1999) A comparison of Notch, Hes and Grg expression during murine embryonic and post-natal development. Cell Mol Biol (Noisy-le-grand) 45:687–708. 38. Heuer H, et al. (2003) Connective tissue growth factor: A novel marker of layer VII neurons in the rat cerebral cortex. Neuroscience 119:43–52. 39. Alcántara S, et al. (1998) Regional and cellular patterns of reelin mRNA expression in the forebrain of the developing and adult mouse. J Neurosci 18:7779–7799. 40. Zhao Y, et al. (1999) Control of hippocampal morphogenesis and neuronal differentiation by the LIM homeobox gene Lhx5. Science 284:1155–1158. 41. Meyer G, Perez-Garcia CG, Abraham H, Caput D (2002) Expression of p73 and Reelin in the developing human cortex. J Neurosci 22:4973–4986.

Other Supporting Information

Dataset S1 (XLS) Dataset S2 (XLS)

Bedogni et al. www.pnas.org/cgi/content/short/1002285107 11 of 11