Supporting Information

McKenna et al. 10.1073/pnas.1504144112 SI Results Primary antibodies used in this study were as follows: chicken Corticothalamic Axons Are Present in the Satb2 cKO Mice. The ex- anti–β-gal (ab9361; Abcam), 1:1,000; rat anti-CTIP2 (ab18465; pression of SATB2 in corticothalamic (Fig. 1 E–H and J) Abcam), 1:200; rabbit anti-CUX1 (sc-13024; Santa Cruz Bio- suggests that it may regulate the development of corticothalamic technology), 1:500; rabbit anti-DARPP32 (ab40801; Abcam), neurons and axons. To examine the corticofugal axons in Satb2 1:200; goat anti-BHLHB5 (sc-6045; Santa Cruz Biotechnology), cKO mice, we used two genetic markers to specifically label PLAP 1:100; rabbit anti-FOG2 (ZFMP2; sc-10755; Santa Cruz Bio- these axons. The Fezf2 allele contains a human PLAP technology), FOXP2 (ab16046; Abcam), 1:1,000; rabbit anti- Fezf2 cDNA that was inserted in the , and labels cortico- GFP (A11122; Invitrogen), 1:1,000; rabbit anti-NFIA (39397; Golli-GFP thalamic and subcerebral axons with PLAP (6). The Active Motif), 1:500; rabbit anti-PLAP (AHP537; Accurate transgene specifically labels corticothalamic and some sub- Chemical), 1:100; rabbit anti-SATB2 (ab34735; Abcam), 1:1,000; cerebral axons (37). lacZ/+ PLAP/+ mouse anti-SATB2 (sc-81376; Santa Cruz Biotechnology), 1:1,000; Compared with control Satb2 ; Fezf2 mice, we found – and rabbit anti-TBR1 (ab31940; Abcam), 1:500. that more Satb2-LacZ labeled axons were present in the thala- + Emx1-Cre Satb2+/+ enhancer Emx1-Cre Satb2lacZ/flox Fezf2PLAP/+ Satb2 cKO For counting of LacZ cells in the ; ; mus of the P4 ; ; ( ) 434-lacZ Emx1-Cre Satb2flox/flox enhancer-434 Satb2 brains (Fig. S6 A and B). This result is consistent with a recent (control) and ; ; ( cKO n = study (25). Intriguingly, fewer Fezf2-PLAP labeled corticothalamic )cortices( 4 for each genotype, collected from three litters), lacZ/flox PLAP/+ axons were present in the P4 Emx1-Cre; Satb2 ; Fezf2 three sections stained with X-Gal were counted from each animal. + + brains (Fig. S6 C and D). Similarly, reduction of Golli-GFP cor- The quantification for the control genotype was 59 ± 8.2 LacZ lacZ/flox ticothalamic axons was observed in P4 Emx1-Cre; Satb2 ; neurons per square millimeter (mean ± SD), and for the Satb2 cKO + Golli-GFP brains. was 23 ± 9.0 LacZ neurons per square millimeter. To determine To further investigate the corticothalamic axons, we compared statistical significance, GraphPad was used to preform an un- + +/+ the GFP axons in Emx1-Cre; Satb2 ; RCE-GFP (control) and paired t test between experimental groups. lacZ/flox Emx1-Cre; Satb2 ; RCE-GFP (Satb2 cKO) brains. Although + GFP corticospinal axons were absent in the Satb2 cKO mice, a Microscopy. Bright-field and epifluorescence images were taken + +/+ careful comparison of GFP axons between Emx1-Cre; Satb2 ; on an Olympus BX51 microscope by using a Q Imaging Retiga lacZ/flox RCE-GFP and Emx1-Cre; Satb2 ; RCE-GFP brains at P24 EXj camera or a Keyence BZ-9000 microscope. Confocal images did not show a significant difference in the corticothalamic in- were obtained on a Leica TCS SP5 or a Zeiss LSM5 confocal nervation patterns. microscope. Images were processed by using Adobe Photoshop E–P Puzzled by this, we investigated Golli-GFP (Fig. S6 ) and CS5 to adjust brightness and contrast. Fezf2-PLAP (Fig. 4) expression in the cortex, and discovered that these genetic markers were no longer expressed in layer 5 Luciferase Assay. Enhancer 434 was amplified from mouse geno- and upper layer 6 neurons in the Satb2 mutant mice. In the mean mic DNA using primers 5′-ATCGCTCGAGCAGGCTGTAGG- time, we observed complementary expanded expression domain ATGGGCAGCAGGAGTTTC-3′ and 5′-ATCGAAGCTTGTA- of the Satb2-LacZ reporter in the deep layer neurons (Fig. 4 and ′ + + ACAAGTCAGGTGAGCAGGCGGTA-3 . The amplified DNA Fig. S6). A comparison of Golli-GFP and Satb2-LacZ axons Satb2lacZ/+ Golli-GFP Emx1-Cre Satb2lacZ/flox was inserted into the SalI site of the luciferase reporter plasmid between P28 ; and ; ; pGL4CP-TK (29) in both orientations. Thus, the enhancer 434 Golli-GFP mice showed that, although some thalamic nuclei + was inserted downstream of the polyadenylation site of the lu- received less innervation from Golli-GFP axons, more Satb2- + LacZ axons were observed in these nuclei, so that the total ciferase gene. + + The SATB2 binding site in the intron of Sox5 was amplified by innervation pattern by the Golli-GFP and Satb2-LacZ axons ′ ′ was not significantly affected in the Satb2 cKO mice (Fig. S6 E– using primers 5 -ATCCACAATGGTCCGCGGGTAACCG-3 + ′ ′ P). The reduced innervation by Golli-GFP axons was most and 5 -TGTAGGAGGTGCCACAGTGCTGGG-3 . The ampli- apparent in the thalamic nuclei targeted by layer 5 cortico- fied sequence was inserted into the BsrG1 sites of the pGL4CP-TK + thalamic axons (Fig. S6 E–P) (46). More Satb2-LacZ cortico- plasmid in both orientations, upstream of the HSV thymidine ki- thalamic axons were observed in these same nuclei (Fig. S6 E–P). nase gene promoter, which drives luciferase . Thus, the corticothalamic axonal defects observed in the Satb2 Neuro-2A cells (American Type Culture Collection) were mutant brains using Golli-GFP, Fezf2-PLAP and Satb2-LacZ transfected with pCAG-Satb2 (gift from Nenad Sestan, Yale axonal reporters were at least partially attributable to the changed University, New Haven, CT) or empty pCAG vector, Renilla reporter expressions. Overall, our axonal analysis suggested that luciferase (pRL-SV40) plasmids (internal control), and empty Satb2 has a more limited role in regulating the development of pGL4CP-TK firefly luciferase reporter plasmids, or pGL4CP-TK corticothalamic axons. reporter plasmids containing enhancer 434 or SATB2 binding site in the Sox5 gene, using Lipofectamine 2000 (Invitrogen). SI Methods β Luciferase activities were assayed 48 h later using the Dual- Retrograde Tracing. The fluorescent tracer CT conjugated with Luciferase System (Promega). Three technical replicates were mea- Alexa 488 or Alexa 594 was injected into the thalamus or pyra- sured for each biological replicate. The ratio of firefly luciferase and midal decussation by using Vevo770 ultrasound-guided injection Renillaluciferaseforeachreplicatewascalculated.InFigs.2E and systems on E17.5 or P2. The brains were collected on P2 or P8 I E and processed for immunohistochemistry. 3 , relative luciferase activities for enhancer 434 (Fig. 2 ) and SATB2 binding site in Sox5 (Fig. 3I) in the presence of Satb2 cDNA are Histochemistry. Immunohistochemistry and PLAP staining for mouse shown relative to the activity in the empty pGL4CP-TK plasmid tissues were performed as previously described (9, 39). LacZ staining measured in the presence of Satb2 cDNA. Error bars represent SD. was performed as previously described (15). All experiments were repeated three times.

McKenna et al. www.pnas.org/cgi/content/short/1504144112 1of8 Fig. S1. (A–C) LacZ staining shows that Satb2-LacZ+ axons are present in the (red star, A), internal capsule (red arrow, A), cerebral peduncle (red arrow, C), and thalamus (red arrow, B). LacZ staining in the cerebral peduncle and thalamus is less intense than in the corpus callosum. (D–K)Im- munostaining shows that the rabbit SATB2 antibody (Abcam) is specific to the SATB2 . (D) SATB2 immunostaining of a P0 WT cortex. (E) No SATB2 lacZ/lacZ flox/+ antibody staining was detected in the Satb2 mice at P0. (F) Low-magnification DAPI image of the control (Satb2 ) brain section shown in G and H. (G) SATB2 immunostaining of the section in F.(H) Higher-magnification image of the boxed area in F and G shows SATB2 staining. (I) DAPI staining of a Satb2 flox/flox cKO (Satb2 ; Emx1-Cre) brain section. (J) SATB2 staining of the same brain section shown in I.(K) Higher-magnification image of the boxed area in I and J shows SATB2 staining. (Scale bars: A–C, 500 μm; D and E, 100 μm; F and I,500μm; G and J, 500 μm; H and K, 100 μm.)

McKenna et al. www.pnas.org/cgi/content/short/1504144112 2of8 Fig. S2. Expressions of upper-layer neuronal and deep-layer neuronal genes were misregulated in Satb2 mutant mice. (A) RNA-seq analysis showed that lacZ/lacZ +/+ 3,129 genes were misregulated (adjusted P < 0.1) in the P0 Satb2 cortices. Compared with Satb2 cortices, at least 15 upper-layer genes (red tick marks) lacZ/lacZ showed reduced expression, and at least 74 deep-layer genes (blue tick marks) showed increased or decreased expression in the Satb2 cortices. lacZ/lacZ +/+ lacZ/lacZ (B–E) TLE4 expression is reduced in the P0 Satb2 cortices. (B and C) Satb2 brains. (D and E) Satb2 brains. (F–I) NFIA expression is reduced in the P0 Satb2lacZ/lacZ brains. (F and G) Satb2+/+ brains. (H and I) Satb2lacZ/lacZ brains. (J and K) DARPP32 expression was reduced in the Satb2lacZ/lacZ brains. (J) DARPP32 staining in Satb2lacZ/+ cortices. (K) Satb2lacZ/lacZ cortices. (Scale bars: B–E,100μm; F–I, 100 μm; J and K,500μm.)

McKenna et al. www.pnas.org/cgi/content/short/1504144112 3of8 Fig. S3. SATB2 regulates Fezf2 expression. (A–J) In situ hybridization showed that Fezf2 mRNA expression was reduced in the E16.5 (A–F) and P0 (G–J) lacZ/lacZ +/+ lacZ/lacZ Satb2 cortices. (A–C, G, and H) Satb2 brains. (D–F, I, and J) Satb2 brains. (K) Immunohistochemistry revealed that β-gal was expressed in cortical +/+ + flox/flox neurons in the P0 control Emx1-Cre; Satb2 ; enhancer 434-lacZ mice. (L) The number of β-gal neurons was reduced in P0 Emx1-Cre; Satb2 ; enhancer +/+ + 434-lacZ mice. (M) Immunohistochemistry for β-gal in the P4 control Emx1-Cre; Satb2 ; enhancer 434-lacZ mice. (N) The number of β-gal neurons was re- flox/flox duced in P4 Emx1-Cre; Satb2 ; enhancer 434-lacZ mice. The enlarged images of the boxed areas in M and N are shown in Fig. 2 F–H, I, and K, respectively. (Scale bars: A–F, 500 μm; G–J, 500 μm; K and L, 100 μm; M and N, 500 μm.)

Fig. S4. Luciferase assays suggest that SATB2 likely directly regulates Fezf2 and Sox5 expression. (A) Cotransfection with pCAG-Satb2 reduced the luciferase activity of the pGL4CP-TK empty plasmid (control). However, cotransfection with pCAG-Satb2 increased the luciferase activity of pGL4CP-TK containing the enhancer 434 sequence in either orientation. (B) Cotransfection with pCAG-Satb2 led to reduction of the luciferase activity of the pGL4CP-TK empty plasmid (control). When the SATB2 binding sequence in the Sox5 intron was cloned into the pGL4CP-TK in either direction, the reduction of the luciferase activity was partially mitigated. The relative luciferase activity for each reporter plasmid in the presence of pCAG-Satb2 was normalized to the activity of the same reporter plasmid in the presence of pCAG plasmid. Error bars indicate SD.

McKenna et al. www.pnas.org/cgi/content/short/1504144112 4of8 Fig. S5. Subcerebral neurons, labeled by high-level CTIP2 expression, were missing in layer 5 and the lateral regions of the in Sox5 cKO mice. +/+ flox/lacZ CTIP2 staining in P4 brains is shown. (A, C, E, G, and I) Sox5 mice. (B, D, F, H, and J) Emx1-Cre; Sox5 mice. (Scale bar: 200 μm.)

McKenna et al. www.pnas.org/cgi/content/short/1504144112 5of8 Fig. S6. Innervation pattern of the thalamus by corticothalamic axons appears not to be significantly changed in the Satb2 mutant mice. (A and B)LacZ + lacZ/flox lacZ/+ staining revealed more Satb2-LacZ axons were present in the thalamus of P4 Emx1-Cre; Satb2 mice (Satb2 cKO, B) than in the Satb2 mice (A). (C and D) + lacZ/flox PALP/+ PLAP staining revealed that fewer Fezf2-PLAP axons were present in the thalamus of P4 Emx1-Cre; Satb2 ; Fezf2 mice (Satb2 cKO, D) than in the lacZ/+ PALP/+ Satb2 Fezf2 mice (C). (E–P) Complementary changes in the innervation patterns of Golli-GFP and Satb2-LacZ–labeled corticothalamic axons in the P28 lacZ/+ lacZ/flox Satb2 cKO mice. Golli-GFP labeling is in green, and Satb2-LacZ staining is in red. (E–G and K–M) Sabt2 ; Golli-GFP mice. (H–J and N–P) Emx1-Cre; Satb2 ; + Golli-GFP mice. Note the reduced corticothalamic innervation by the Golli-GFP axons in the Satb2 cKO mice (compare arrows in H to E and in N to K), and the increased innervation by the Satb2-LacZ+ axons (compare arrows in I to F and in O to L). The arrows in E–J point to the laterodorsal (LD) thalamic nuclei, and the arrows in K–P point to the posterior (Po) thalamic nuclei. Both are targets of layer 5 corticothalamic axons in WT mice. (Scale bars: 500 μm.)

McKenna et al. www.pnas.org/cgi/content/short/1504144112 6of8 Table S1. Deep-layer genes based on in situ hybridization data in Allen Brain Atlas that show misregulated expression in the lacZ/lacZ Satb2 cortices lacZ/lacZ Gene name Changes in Satb2 cortices P value

Grm2 0.20529751 6.61E-75 Cdh6 0.27675038 1.91E-24 Npy 0.16025512 3.66E-84 Dkk3 0.30507669 3.79E-32 Tgfbr1 0.30654068 6.12E-27 0.35832214 7.73E-33 Sstr2 0.37881725 2.49E-12 Otx1 0.39990856 1.54E-15 Scg2 0.39992902 5.15E-08 Foxp2 0.40370881 6.72E-08 Nfe2l3 0.43725345 2.54E-13 Pdzd2 0.46468576 1.71E-16 Kcnab1 0.47601412 1.13E-05 Cyp26b1 0.47704768 2.34E-08 Nr4a3 0.48352674 1.15E-11 Ppp1r1b 0.48854776 4.84E-14 Lrrtm1 0.50806766 2.01E-17 Lypd1 0.5307642 6.19E-06 Grm3 0.53273906 8.76E-10 Fosl2 0.54704973 1.65E-10 Kit 0.58440677 2.78E-06 Nfia 0.584763 6.28E-07 Sfrp2 0.6428176 3.61E-04 Hivep2 0.64363187 3.77E-08 Sox5 0.65492649 1.29E-05 Fezf2 0.65668887 9.96E-07 Tle4 0.66497135 6.81E-06 Chrna4 0.70498442 1.32E-03 Tox 0.72285593 2.51E-03 Ptpru 0.73107426 4.51E-03 Cdh11 0.74388182 1.14E-02 Adnp2 0.74487771 4.70E-03 Atf4 0.7648128 2.12E-03 Robo1 0.7853079 3.78E-03 Kctd12 0.7905371 2.32E-03 Wnt7b 0.79813825 1.50E-03 Apba1 1.23865013 8.80E-03 Ldb2 1.25899226 1.27E-02 Atp1a1 1.29657591 4.21E-03 Efnb3 1.30906908 7.00E-04 Plxnd1 1.31274827 6.25E-04 S100a10 1.31547043 1.39E-02 Slc32a1 1.31927601 5.05E-03 Grm5 1.37255398 2.18E-04 Tmed3 1.38418955 1.32E-02 Reln 1.39426911 3.93E-05 Clu 1.40642198 1.64E-03 Dner 1.41103694 4.54E-05 Cpne2 1.43475201 6.17E-03 Vat1l 1.47598159 1.94E-03 Grin3a 1.53615672 1.13E-03 Doc2b 1.54698299 7.32E-05 Gria3 1.58524317 2.99E-03 Mgst3 1.62468235 2.25E-05 Zdhhc2 1.63901717 2.97E-03 Gls 1.74580402 9.65E-06 Ephb1 1.755501 2.53E-10 Cadm1 2.27607807 2.16E-16 Grik2 2.28630348 2.17E-08 Glra2 2.28872138 7.77E-11 Sema3c 2.3761516 6.57E-13

McKenna et al. www.pnas.org/cgi/content/short/1504144112 7of8 Table S1. Cont. Gene name Changes in Satb2lacZ/lacZ cortices P value

Bcl11b 2.53050009 1.31E-30 Pex5l 2.55438504 1.48E-07 Tmem163 2.76022443 2.19E-29 Kitl 2.90448579 3.75E-20 Slc16a2 2.92383611 1.58E-26 Cntn6 3.02923239 1.82E-08 Crtac1 3.3299394 5.61E-19 Cdh7 3.56685025 2.33E-04 Gda 3.59780471 1.64E-16 Ntng1 3.75020557 1.09E-10 Nov 4.54107996 1.89E-27 Crym 5.2450782 3.93E-41 Alcam 5.26687303 2.41E-39

Table S2. Upper-layer genes based on Allen Brain Atlas that lacZ/lacZ show decreased expression in the Satb2 cortices lacZ/lacZ Gene name Changes in Satb2 cortices P value

Dtx4 0.27289644 3.25E-60 Ephb6 0.45689849 6.69E-23 Sema7a 0.47282016 8.17E-23 Cacna2d1 0.48087673 2.56E-09 Zfp462 0.48536269 9.02E-18 Bhlhe40 0.49070703 1.92E-07 Cux1 0.4971219 1.19E-14 Odz1 0.53998315 5.06E-05 Trpc4 0.57444866 9.36E-06 Pvrl3 0.58411498 1.10E-03 Lhx2 0.63835929 3.90E-09 Cux2 0.72810086 1.32E-05 Mef2c 0.73833696 5.83E-05 Ntng2 0.75951106 1.10E-03 Mdga1 0.8267283 1.45E-02

McKenna et al. www.pnas.org/cgi/content/short/1504144112 8of8