SUPPLEMENTARY MATERIALS AND METHODS

Bioinformatic analysis of Allen Brain Atlas data. All ISH data in the Allen

Brain Atlas (ABA, http://mouse.brain-map.org, Lein et al., 2007) is spatially registered

to a common 3D atlas space derived from the Allen Reference Atlas (ARA,

http://mouse.brain-map.org/atlas/index.html, Dong, 2007) using previously described

image processing algorithms (Ng et al., 2007). Thus, 3D regions of interest (ROI) can be

identified in reference atlas space and projected onto the ISH data for analysis. To

quantify habenula enrichment, three ROIs were used: Habenula, consisting of both the

medial and lateral habenula on one hemisphere as defined in the ARA. Thalamus, as

defined in the ARA with the exclusion of the medial and lateral habenula. Cortex, defined

as the isocortex region of the ARA, further bounded in the anterior-posterior axis by

Bregma -2mm to 0mm and along the medial-lateral axis by Lateral 1 to 3mm.

Expression signal extraction from the ISH data is performed using morphological

filtering and adaptive thresholding techniques resulting in an expression mask for each

ISH image which classifies each pixel as either expressing or non-expressing (Ng et al.,

2007). From the expressing pixels in the ROI, the expression intensity is then used to calculate a measure of “expression energy”, E(R) defined for a region R as:

∑ ( )× (pIpM ) ()RE = ∈Rp R

where p is a image pixel that intersects region R, |R| is the total number of pixels that

intersect R, M(p) is the expression segmentation mask which is either 1 (“expressing”

pixel) or 0 (“non expressing” pixel) and I(p) is the grayscale value of the ISH image

intensity defined as Gray = 0.3*Red + 0.59*Green + 0.11*Blue. The advantages of defining expression energy in this way is that it can be robustly computed over all brain

regions; it is easily normalized to account for different image resolution and/or section

sampling frequency; it is amenable to smoothing for noise suppression; and it combines

features of expression intensity and expression density into a single measurement.

For each of the ROI listed above an expression energy value was computed and

Habenula/Cortex and Habenula/Thalamus ratios were computed as an informatics

derived measure of enrichment. In rare instances the bioinformatic analysis failed to

correctly map the habenula to the reference atlas due to the location or quality of the

section (Robo3); this occurred only for data sets in the sagittal plane, in which the

habenula is represented in only one or two sections. Such cases were discarded in the

manual curation of the results.

Statistical comparision of microarray and bioinformatic data for habenula-

enriched transcripts. We identified 112 unique transcripts in the microarray data set

which had the highly enriched expression in the habenula compared to the thalamus and

cortex. For these comparisions, habenula expression was at least 10-fold greater than the

mean expression in the cortex/thalamus. Results were also filtered for transcripts which

exhibited at least half of the target scaled expression value of 500, and had a present “P”

call in both habenula samples. Of these 112 transcripts, 91 had extractable data in the

ABA. Of these, ABA data showed tthat 42 had enriched expression in the adult

habenula, and 30 had detectable expression which was not enriched in the habenula, for a total of 72 probes with detectable expression. In addition, 19 had habenula expression below the reliable threshold of detection (Figure 3A). We also selected 88 control transcripts from the microarray experiment which had equal expression in the habenula and thalamus/cortex (Figure 3B). Of these, only 3 showed increased habenula expression

in the analysis of the ABA data, from among 52 which had detectable expression. To compute the statistical significance of these results, we compared the empirical results

with a null hypothesis of randomly selecting probes from set U, all ABA probes for

which habenula energy > 1.0 where |U| = 10108. Within U, 1654 probes have habenula

enrichment > 2-fold. Since U is large, we can approximate the probability of obtaining X

probes with > 2-fold enrichment out of random selection of N by a binomial distribution with p = (1654/10108) = 0.164. Performing this binomial test we get a p-value of 4.44 x

10-16 (highly unlikely) for obtaining greater than 42 enriched probes from a random

selection of 72 and p-value of 0.98 (highly likely) for obtaining greater than 3 enriched

probes from a random selection of 52.

ADDITIONAL REFERENCES

Dong H-W (2007) Allen reference atlas : a digital color brain atlas of the C57Black/6J male mouse. Hoboken, N.J.Chichester: Wiley; John Wiley distributor. Lein ES, Hawrylycz MJ, Ao N, Ayres M, Bensinger A, Bernard A, Boe AF, Boguski MS, Brockway KS, Byrnes EJ, Chen L, Chen TM, Chin MC, Chong J, Crook BE, Czaplinska A, Dang CN, Datta S, Dee NR, Desaki AL, Desta T, Diep E, Dolbeare TA, Donelan MJ, Dong HW, Dougherty JG, Duncan BJ, Ebbert AJ, Eichele G, Estin LK, Faber C, Facer BA, Fields R, Fischer SR, Fliss TP, Frensley C, Gates SN, Glattfelder KJ, Halverson KR, Hart MR, Hohmann JG, Howell MP, Jeung DP, Johnson RA, Karr PT, Kawal R, Kidney JM, Knapik RH, Kuan CL, Lake JH, Laramee AR, Larsen KD, Lau C, Lemon TA, Liang AJ, Liu Y, Luong LT, Michaels J, Morgan JJ, Morgan RJ, Mortrud MT, Mosqueda NF, Ng LL, Ng R, Orta GJ, Overly CC, Pak TH, Parry SE, Pathak SD, Pearson OC, Puchalski RB, Riley ZL, Rockett HR, Rowland SA, Royall JJ, Ruiz MJ, Sarno NR, Schaffnit K, Shapovalova NV, Sivisay T, Slaughterbeck CR, Smith SC, Smith KA, Smith BI, Sodt AJ, Stewart NN, Stumpf KR, Sunkin SM, Sutram M, Tam A, Teemer CD, Thaller C, Thompson CL, Varnam LR, Visel A, Whitlock RM, Wohnoutka PE, Wolkey CK, Wong VY, Wood M, et al. (2007) Genome-wide atlas of gene expression in the adult mouse brain. Nature 445:168-176. Ng L, Pathak SD, Kuan C, Lau C, Dong H, Sodt A, Dang C, Avants B, Yushkevich P, Gee JC, Haynor D, Lein E, Jones A, Hawrylycz M (2007) Neuroinformatics for genome-wide 3D gene expression mapping in the mouse brain. IEEE/ACM Trans Comput Biol Bioinform 4:382-393.

PCR generated probes Gene mRNA ID Forward primer Reverse primer Gpr151 NM_181543 394F 1094R Kctd8 NM_175519 1202F 1902R Rtn4rl2 NM_199223 102F 802R Nr4a2 NM_013613 647F 1347R Plch2 NM_175556 3549F 4249R Kcnma1 NM_010610 2128F 2828R Etv1 NM_007960 1185F 1885R Dcc NM_007831 924F 1624R

Other probes Gene Reference Kcnip1 Open Biosystems, BC034241 Neurod1 (Eng et al., 2004) Nrp2 (Loes et al., 2001) Pou4f1 (Eng et al., 2004) Tac1 (Xu et al., 2008)

Supplementary Table 1. In situ hybridization probes.

Eng, S. R., Lanier, J., Fedtsova, N. and Turner, E. E. (2004). Coordinated regulation of gene expression by Brn3a in developing sensory ganglia. Development 131, 3859-70. Loes, S., Kettunen, P., Kvinnsland, I. H., Taniguchi, M., Fujisawa, H. and Luukko, K. (2001). Expression of class 3 semaphorins and neuropilin receptors in the developing mouse tooth. Mech Dev 101, 191-4. Xu, Y., Lopes, C., Qian, Y., Liu, Y., Cheng, L., Goulding, M., Turner, E. E., Lima, D. and Ma, Q. (2008). Tlx1 and Tlx3 coordinate specification of dorsal horn pain-modulatory peptidergic neurons. J Neurosci 28, 4037-46.

Bioinfomatic analysis Visual analysis

ABA Gene Section Hab Ctx Thal Ctx Thal Dors Vent ID Gene name symbol1 plane En’gy En’gy En’gy Ratio Ratio Hab MH MH LH Ctx Thal 74989 cholinergic receptor, nicotinic, alpha polypeptide 3 Chrna3 coronal 10.12 0.01 0.12 1059 84313000 17839 nescient helix loop helix 2 Nhlh2 coronal 8.10 0.03 0.35 257 23331100 17927 natriuretic peptide receptor 1 Npr1 coronal 7.20 0.03 0.28 255 26313000 88694 G protein-coupled receptor 151 Gpr151 coronal 15.37 0.09 0.61 179 25303200 72177 cholinergic receptor, nicotinic, beta polypeptide 4 Chrnb4 coronal 4.58 0.03 0.06 177 80313000 88861 tetraspanin 18 Tspan18 coronal 8.89 0.06 0.27 140 32313010 14559 growth factor receptor bound protein 10 Grb10 coronal 2.49 0.02 0.23 131 11111101 65942 transmembrane protein 16A Tmem16a coronal 3.15 0.03 0.06 123 52222000 138282 TIGR MGI TC1563370 TC156337 0 coronal 6.48 0.06 1.17 1156222011 18525 protein kinase C, theta Prkcq coronal 12.90 0.11 0.36 112 36313110 41806 RIKEN cDNA 1810009M01 gene 1810009 M01Rik coronal 6.15 0.05 0.22 112 28222001 85128 sterile alpha motif domain containing 14 Samd14 coronal 15.65 0.14 2.07 1098333111 69992 adhesion molecule with Ig like domain 2 Amigo2 coronal 10.40 0.10 0.89 102 12313111 11926 fatty acid binding protein 7, brain Fabp7 coronal 5.67 0.06 0.39 94 14331000 17954 neuropilin 2 Nrp2 sagittal9318978515333.d.10 14.0.0. n 15337 5 hydroxytryptamine (serotonin) receptor 4 Htr4 coronal 1.46 0.02 0.06 85 24101000 93438 WAP, follistatin/kazal, immunoglobulin, kunitz and netrin domain containing 2 Wfikkn2 sagittal6710288027313.d.10 7.0.0. n 18759 POU domain, class 4, transcription factor 1; Brn3a Pou4f1 coronal 14.16 0.19 0.20 75 69333100 126225 RIKEN cDNA B930011P16 gene B930011P coronal 3.23 0.05 0.09 69 35222000 16Rik 16145 Iroquois related homeobox 2 Irx2 coronal 3.36 0.05 0.12 68 29222100 20270 solute carrier family 18, member 3; VAChT Slc18a3 coronal 5.92 0.09 0.10 67 62313000 84078 cytosolic ovarian carcinoma antigen 1 Cova1 coronal 4.71 0.07 0.57 638222000 45627 Tp53rk binding protein Tprkb sagittal0903116020111.d.00 2.0.0. n 22082 Vav2 oncogene Vav2 coronal 13.76 0.23 0.17 59 80333010 20380 synuclein, gamma Sncg coronal 14.85 0.26 0.75 57 20333211 52221 coiled-coil domain containing 46 Ccdc46 sagittal230221566111.d.11 1.0.0. n 18850 protein kinase, cAMP dependent regulatory, type II alpha Prkar2a coronal 6.10 0.11 0.12 55 51313000 34001 plexin C1 Plxnc1 coronal 1.33 0.03 0.04 51 30111000 86747 early B-cell factor 4 Ebf4 coronal 1.69 0.03 0.05 49 36111000 21097 tachykinin receptor 1 Tacr1 coronal 3.27 0.07 0.14 48 24303010 18157 opioid receptor, mu 1 Oprm1 coronal 3.88 0.08 0.71 475333001 37970 synaptotagmin IX Syt9 coronal 20.31 0.45 4.72 454333002 33815 synaptotagmin VI Syt6 coronal 3.50 0.08 0.06 44 60222010 43840 DEAD (Asp-Glu-Ala-Asp) box polypeptide 59 Ddx59 sagittal4803054332110.d.00 1.0.0. n 15339 5-hydroxytryptamine (serotonin) receptor 5B Htr5b coronal 4.01 0.09 0.12 43 33313000 23680 laminin gamma 3 Lamc3 sagittal9028484225333.d.00 11.0.0. n 89268 signal-induced proliferation- associated 1 like 2 Sipa1l2 coronal 4.98 0.12 0.36 42 14222000 42150 isochorismatase domain containing 1 Isoc1 coronal 5.96 0.15 0.64 419313110 16743 low density lipoprotein receptor- related protein 5 Lrp5 coronal 2.00 0.05 0.07 41 29111000 41812 cubilin (intrinsic factor-cobalamin receptor) Cubn sagittal3803044035220.d.00 1.0.0. n 92853 tetraspanin 12 Tspan12 coronal 1.02 0.03 0.08 39 13111010 89811 prokineticin receptor 2 Prokr2 coronal 3.20 0.08 0.55 386000111 16364 klotho Kl coronal 7.70 0.21 0.80 37 10111011 72311 semaphorin 3D Sema3d coronal 1.59 0.04 0.07 37 23000100 89217 RIKEN cDNA C230040D10 gene C230040 D10Rik coronal 6.65 0.19 0.09 35 73313000 21095 tachykinin 2, substance K Tac2 coronal 23.97 0.70 0.60 34 40333020 72205 cholinergic receptor, nicotinic, beta polypeptide 3 Chrnb3 coronal 7.26 0.22 0.21 33 34313010 86337 transient receptor potential cation channel, subfamily M, member 3 Trpm3 sagittal512075329111.d.10 6.0.0. n 11355 AE binding protein 1 Aebp1 sagittal811264326222.d.00 3.0.0. n 35270 chemokine (C-X-C motif) ligand 13 Cxcl13 coronal 1.09 0.03 0.13 318000200 16930 monoamine oxidase A Maoa coronal 2.00 0.06 0.20 31 10111111 48542 zinc fingerprotein 618 Zfp618 coronal 1.17 0.04 0.07 31 17111000 35803 polyamine modulated factor 1 binding protein 1 Pmfbp1 sagittal5205103115222.d.00 1.0.0. n 15021 Hedgehog-interacting protein Hhip coronal 5.51 0.18 0.16 31 34333010 30013 large tumor suppressor 2 Lats2 coronal 3.31 0.11 0.49 297111010 21567 transforming growth factor, beta 2 Tgfb2 coronal 4.86 0.17 0.41 29 12111011 45152 Park2 co-regulated Pacrg coronal 2.80 0.10 0.21 29 13111010 68436 testis expressed gene 15 Tex15 sagittal2915242918222.d.00 4.0.0. n 21708 tankyrase, TRF1-interacting ankyrin-related ADP-ribose polymerase Tnks sagittal7217462810111.d.11 4.0.0. n 41901 RIKEN cDNA 0610011I04 gene 0610011I 04Rik coronal 6.24 0.23 0.70 289222001 33162 procollagen, type V, alpha 3 Col5a3 coronal 2.66 0.10 0.59 285222010 40708 SPARC related modular calcium binding 1 Smoc1 coronal 3.65 0.14 0.36 26 10303011 16281 potassium voltage-gated channel, Shal-related family, member 2 Kcnd2 coronal 3.45 0.15 0.21 23 17222010 78444 EF hand calcium binding protein 2 Efcbp2 coronal 30.64 1.35 7.60 234333111 88742 transmembrane protein 20 Tmem20 sagittal9809042344101.d.00 1.0.0. n 50159 homeobox only domain Hod coronal 1.51 0.07 0.13 22 12212000 42104 transmembrane 4 L six family member 20 Tm4sf20 sagittal954421228111.d.11 9.0.1. n 14542 G protein-coupled receptor 56 Gpr56 coronal 5.93 0.27 1.11 225111000 85326 expressed sequence AW456874 AW45687 4 coronal 2.85 0.13 0.05 22 61212000 14361 glial cell line derived neurotrophic factor family receptor alpha 1 Gfra1 coronal 21.55 0.97 2.32 229333110 86982 coenzyme Q3 homolog, methyltransferase (yeast) Coq3 sagittal121558215111.d.11 3.0.0. n 42658 coiled-coil domain containing 109B Ccdc109b coronal 2.81 0.13 0.53 215222001 37653 poly(rC) binding protein 3 Pcbp3 coronal 2.38 0.12 0.33 207111001 35618 thioredoxin interacting protein Txnip sagittal460731205111.d.00 1.0.0. n 84303 potassium voltage-gated channel, subfamily H (eag- related), member 8 Kcnh8 coronal 3.50 0.18 0.30 20 12111100 89032 potassium channel tetramerisation domain containing 8 Kctd8 coronal 7.55 0.39 0.27 19 28222000 11616 aquaporin 4 Aqp4 coronal 15.86 0.82 3.08 195222111 107762 RIKEN cDNA C530008M17 C530008 gene M17Rik coronal 5.47 0.29 0.95 196222101 84435 SH3 domain binding glutamic acid-rich protein like 2 Sh3bgrl2 coronal 5.47 0.29 0.68 198333111 27849 tripartite motif-containing 36 Trim36 coronal 4.91 0.26 0.20 19 25222010 41954 transmembrane emp24 domain containing 3 Tmed3 coronal 2.48 0.13 0.28 189111111 19866 ribosomal protein S5 Rps5 coronal 1.40 0.08 0.26 185111000 22171 wingless related MMTV integration site 2b Wnt2b sagittal3608141810111.d.10 1.0.0. n 40873 neuroglobin Ngb coronal 1.55 0.09 0.32 175111110 17457 Musashi homolog 1(Drosophila) Msi1 sagittal721631179111.d.00 2.0.0. n 84142 cDNA sequence AF529169 AF529169 coronal 6.93 0.41 0.13 17 54313010 85407 leucine rich repeat containing 3B Lrrc3b coronal 7.04 0.42 0.36 17 19333010 129444 zinc finger homeobox 2, antisense Zfhx2as sagittal5640491713222.d.11 6.0.0. n 14356 glial fibrillary acidic protein Gfap coronal 8.71 0.53 1.64 175222000 17997 nucleoredoxin Nxn coronal 1.86 0.11 0.12 16 16111100 43262 RIKEN cDNA 3632451O06 gene 3632451O 06Rik coronal 12.54 0.78 1.83 167333211 52108 fatty acid desaturase 1 Fads1 sagittal195902169111.d.11 9.0.1. n 106742 RIKEN cDNA 3110047P20 gene 3110047P 20Rik coronal 22.27 1.44 0.68 15 33333311 88859 leucine rich repeat containing 55 Lrrc55 coronal 19.63 1.31 0.30 15 65333110 106487 RIKEN cDNA B230216G23 gene B230216 G23Rik sagittal4610071522212.d.00 1.0.0. n 14146 frizzled homolog 8 (Drosophila) Fzd8 sagittal793209154111.d.11 4.0.1. n 105960 kallikrein 14 Klk14 sagittal0021111527222.d.00 3.0.0. n 16611 leukocyte cell derived chemotaxin 1 Lect1 sagittal531125146111.d.10 1.0.0. n 17779 neurogenic differentiation 1 Neurod1 coronal 5.39 0.38 0.17 14 31222010 149717 RIKEN cDNA 2610304F08 gene 2610304F (non-RefSeq) 08Rik* sagittal7655401419202.d.00 7.0.0. n 15304 syndecan 2 Sdc2 coronal 3.99 0.29 0.84 145000111 108453 poly (ADP-ribose) polymerase family, member 4 Parp4 sagittal0615121417111.d.00 2.0.0. n 88631 G protein-coupled receptor 156 Gpr156 coronal 1.48 0.11 0.14 14 10111000 43359 potassium channel tetramerisation domain containing 4 Kctd4 coronal 7.03 0.52 0.97 137111010 85483 spermatogenesis associated 13 Spata13 coronal 1.18 0.09 0.22 135111010 52729 aarF domain containing kinase 4 Adck4 coronal 4.59 0.36 0.38 13 12111010 45525 alanyl-tRNA synthetase domain Aarsd1 sagittal542061134222.d.11 2.0.0. n containing 1 92784 synaptotagmin-like 1 Sytl1 sagittal4351371317222.d.00 6.0.0. n 86464 RIKEN cDNA 9630058J23 gene 9630058J 23Rik sagittal8864811210111.d.00 7.0.0. n 23868 Wnt inhibitory factor 1 Wif1 coronal 2.93 0.24 0.14 12 21222000 106859 amyloid beta (A4) precursor protein binding, family A, member 1 Apba1 coronal 10.75 0.88 1.03 12 10333111 92622 signal peptide, CUB domain, EGF-like 3 Scube3 coronal 2.53 0.21 0.60 124222011 12349 cadherin 8 Cdh8 coronal 2.66 0.22 0.41 127111110 42791 acyl-Coenzyme A dehydrogenase family, member 8 Acad8 sagittal2569511216111.d.00 8.0.0. n 12011 B-cell translocation gene 1, anti- proliferative Btg1 coronal 6.12 0.53 0.59 12 10221110 17878 neuronatin Nnat coronal 28.16 2.49 5.42 115333122 18282 pre B-cell leukemia transcription factor 3 Pbx3 coronal 6.00 0.54 0.75 118111110 30267 regulator of G-protein signaling 6 Rgs6 coronal 1.83 0.17 0.20 119111010 32926 mitogen-activated protein kinase kinase kinase 6 Map3k6 sagittal3160401116333.d.00 6.0.0. n 21582 tyrosine hydroxylase Th coronal 1.85 0.17 0.22 118212010 17026 methyl CpG binding protein 2 Mecp2 sagittal272236106111.d.11 2.0.0. n 84444 phosphoribosyl pyrophosphate synthetase-associated protein 2 Prpsap2 sagittal215056109111.d.10 5.0.0. n 80269 ankyrin repeat and SOCS box- containing protein 14 Asb14 sagittal531530105111.d.00 1.0.0. n 42930 catenin beta interacting protein 1 Ctnnbip1 sagittal056931105313.d.01 7.0.1. n 41885 sclerostin domain containing 1 Sostdc1 coronal 9.47 0.93 1.46 106222111 82865 transmembrane protein 47 Tmem47 coronal 5.43 0.53 1.33 104111111 88783 similar to hypothetical protein LOC2409 MGC48998 06 sagittal073060105111.d.11 3.0.0. n 46978 regulatory factor X, 4 (influences Rfx4 coronal 1.11 0.11 0.12 109111000 HLA class II expression) 26125 dual adaptor for phosphotyrosine and 3-phosphoinositides 1 Dapp1 sagittal062124109111.d.00 2.0.0. n

Supplementary Table 2. Bioinformatic and visual analysis of habenula enriched genes. Expression energy was calculated for the habenula (medial + lateral), thalamus and cerebral cortex for approximately 20,000 transcripts in the ABA data set, as described in methods. Habenula enrichment was calculated as a ratio of habenula/thalamus energy and habenula/cortex energy. Thresholds for habenula/cortex > 10 and habenula/thalamus > 4 were applied, as well as a minimum habenula energy of 1.0, which corresponded to the approximate level of visual detection of a signal above background. The ISH expression patterns of the resulting set of 306 transcripts were visually inspected in the ABA. Of these transcripts 98 were below the threshold of reliable detection,and in 78 cases expression was observed in the choroid plexus or cells, presumably glia, lining the ventricle. For the remaining 130 transcripts, expression in the total habeula, and the dorsal medial, ventral medial, and lateral habenula, thalamus and cortex was scored on a 0-3 scale. Specific expression in the lateral habenula could not be clearly determined for data series in the sagital plane (n.d.), because the lateral habenula did not appear reliably in these sections.

1Hyperlinked to ABA gene expression data.

SUPPLEMENTARY TABLES

Expression value, A/P call Mean ratio HT/ HB/ HB/ Probe set Gene Title Gene Symbol Het1 KO1 Het2 KO2 KO CTX Thal 1429667_at POU domain, class 4, transcription factor 1; Brn3a Pou4f1 12886 P 118 P 6208 P 182 P 64 180 99 1441495_at Potassium channel tetramerisation domain containing 8 Kctd8 99 P 2 A 422 P 22 A 22 23 8.3 1422196_at 5-hydroxytryptamine (serotonin) receptor 5B Htr5b 242 P 6 A 305 P 34 A 14 69 11 1436474_at Mannose receptor-like precursor Mrcl* 175 A 11 A 135 A 12 A 14 35 6.9 1419148_at Advillin Avil* 143 P 13 A 85 A 6 A 12 17 8.8 1417788_at Synuclein, gamma Sncg 257 P 22 P 616 P 58 P 11 13 7.4 1457555_at G protein-coupled receptor 151 Gpr151 1971 P 209 P 2027 P 248 P 8.8 51 34 1427383_at Iroquois related homeobox 6 Irx6 204 P 13 A 362 P 65 A 7.3 8.4 5.7 1460482_at RIKEN cDNA 3110047P20 gene 3110047P20Rik 13229 P 2801 P 11756 P 2443 P 4.8 188 7.6 1416785_at Kv channel-interacting protein 1 Kcnip1 581 P 116 P 1641 P 360 P 4.7 3.3 1.9 1439573_at Reticulon 4 receptor-like 2 Rtn4rl2 254 P 72 P 825 P 160 P 4.7 0.7 1.7 1420901_a_at Hexokinase 1 Hk1 275 P 109 P 1407 P 261 P 4.5 1.0 0.7 1450044_at Frizzled homolog 7 Fzd7 294 P 51 P 251 P 70 P 4.5 1.5 1.7 1457008_at Cholinergic receptor, nicotinic, beta polypeptide 4 Chrnb4 533 P 163 P 601 P 103 A 4.3 28 14 1457440_at Somatostatin receptor 4 Sstr4 822 P 177 P 434 P 121 P 4.2 14 2.9 1442115_at RIKEN cDNA 9430028L06 gene 9430028L06Rik 218 P 57 P 341 P 79 P 4.1 11 3.9 1455034_at Nuclear receptor subfamily 4, group A, member 2; Nurr1 Nr4a2 7300 P 1902 P 4452 P 1297 P 3.7 8.1 22 1422825_at Cocaine and amphetamine regulated transcript Cart 518 P 154 A 959 P 266 P 3.5 33 5.1 1435125_at Expressed sequence BB120497 BB120497 350 P 104 P 316 P 101 P 3.2 12 25 1436577_at Cdc42 guanine nucleotide exchange factor (GEF) 9 Arhgef9 118 P 64 A 702 P 199 P 3.1 1.1 1.0 1436335_at Phospholipase C, eta 2; Phospholipase C-like 4; Plcl4 Plch2 1524 P 442 P 2022 P 703 P 3.1 6.3 4.5 1453062_at RIKEN cDNA A930026I22 A930026I22Rik 1656 P 577 P 1173 P 337 P 3.1 9.4 3.8 1434785_at Calcium channel, voltage-dependent, gamma subunit 5 Cacng5 396 P 151 P 546 P 155 P 3.1 1.4 2.5 1423258_at synaptotagmin IX Syt9 1921 P 788 P 2744 P 745 P 3.0 9.7 3.4 1445774_at Potassium large conductance calcium-activated channel, subfamily M, alpha member 1 Kcnma1 2315 P 704 P 849 P 378 P 2.9 63 25 1448123_s_at Transforming growth factor, beta induced Tgfbi 398 P 125 P 313 P 127 P 2.8 7.6 5.7 1423475_at Cyclin M2 Cnnm2 471 P 221 P 620 P 178 P 2.7 4.9 3.0 1441185_at Musashi homolog 2 Msi2 3230 P 1067 P 1364 P 637 P 2.7 32 34 1425777_at Calcium channel, voltage-dependent, beta 1 subunit Cacnb1 252 P 107 P 898 P 323 P 2.7 1.6 1.1 1452010_at Cholinergic receptor, nicotinic, alpha polypeptide 3 Chrna3 1203 P 523 P 2408 P 835 P 2.7 45 3.9 1434908_at Expressed sequence AI480556 AI480556 390 P 201 P 810 P 256 P 2.6 0.9 1.5 1421889_a_at Amyloid beta (A4) precursor-like protein 2 Aplp2 498 P 226 P 2891 P 1089 P 2.6 1.3 1.0 1448656_at Calcium channel, voltage-dependent, beta 3 subunit Cacnb3 5191 P 2260 P 8193 P 3118 P 2.5 2.1 3.3 1422477_at Cdk5 and Abl enzyme substrate 1 /// similar to Cdk5 and Abl enzyme substrate 1 Cables1 1230 P 484 P 1119 P 464 P 2.5 4.2 3.8 1428848_a_at Microtubule-actin crosslinking factor 1 Macf1 894 P 437 P 3498 P 1349 P 2.5 1.0 2.4 1434755_at Coronin, actin binding protein, 2B Coro2b 656 P 295 P 1388 P 544 P 2.4 0.9 2.6 1427713_x_at POU domain, class 2, transcription factor 2 Pou2f2 385 P 117 P 372 P 197 P 2.4 7.7 1.1 1426413_at Neurogenic differentiation 1 Neurod1 7968 P 3243 P 4988 P 2211 P 2.4 2.7 12 1423430_at MYB binding protein (P160) 1a Mybbp1a 254 P 187 P 1054 P 365 P 2.4 0.5 1.2 1421276_a_at Dystonin Dst 349 P 124 M 965 P 432 P 2.4 1.0 1.5 1429440_at RIKEN cDNA 1810041L15 gene 1810041L15Rik 1512 P 825 P 2002 P 668 P 2.4 18 1.7 1425425_a_at Wnt inhibitory factor 1 Wif1 756 P 294 P 480 P 238 P 2.3 80 2.5 1449125_at Tumor necrosis factor, alpha-induced protein 8-like 1 Tnfaip8l1 354 P 211 P 669 P 232 P 2.3 1.4 1.2 1438241_at RGM domain family, member A Rgma 875 P 462 P 2148 P 850 P 2.3 3.1 1.8 1422607_at Ets variant gene 1; ER81 Etv1 9202 P 3665 P 7438 P 3639 P 2.3 11 19 1450339_a_at B-cell leukemia/lymphoma 11B Bcl11b 3266 P 1106 P 1853 P 1149 P 2.3 0.3 6.0 1426044_a_at Protein kinase C, theta Prkcq 656 P 274 A 571 P 268 A 2.3 31 4.5 1426348_at Procollagen, type IV, alpha 1 Col4a1 450 P 196 P 883 P 393 P 2.3 1.2 1.5 1458394_at Solute carrier family 6 (neurotransmitter transporter), member 17 Slc6a17 275 P 121 A 816 P 364 P 2.2 0.9 1.5 1452913_at Purkinje cell protein 4-like 1 Pcp4l1 90 P 41 A 209 P 93 P 2.2 5.8 1.1 1419341_at Eph receptor A8 Epha8 846 P 549 P 1127 P 338 P 2.2 24 3.5 1438700_at Formin binding protein 4 Fnbp4 228 P 96 P 293 P 139 P 2.2 3.2 6.6 1447602_x_at Sulfatase 2 Sulf2 6679 P 2874 P 4863 P 2335 P 2.2 10 2.8 1460214_at Purkinje cell protein 4 Pcp4 7900 P 5167 P 14051 P 4808 P 2.2 8.8 2.1 1423308_at Trans-golgi network protein Tgoln1 126 P 47 P 394 P 190 P 2.2 1.2 2.1 1427414_at Protein kinase, cAMP dependent regulatory, type II alpha Prkar2a 142 P 73 P 400 P 176 P 2.2 0.8 0.8 1460250_at Sclerostin domain containing 1 Sostdc1 588 P 305 P 839 P 350 P 2.2 15 9.7 1448348_at GPI-anchored membrane protein 1 Gpiap1 172 P 71 A 883 P 417 P 2.2 0.6 1.3 1424768_at Caldesmon 1 Cald1 101 P 48 P 514 P 237 P 2.2 1.0 0.8 1447707_s_at Phosphodiesterase 2A, cGMP- stimulated Pde2a 8542 P 4029 P 6261 P 2898 P 2.1 5.4 9.2 1423630_at Cytoglobin Cygb 242 P 101 P 1208 P 585 P 2.1 3.3 2.3 1425732_a_at Max interacting protein 1 Mxi1 165 P 94 A 810 P 369 P 2.1 0.9 1.7 1419118_at RIKEN cDNA 2900093B09 gene 2900093B09Rik 2401 P 1036 P 2410 P 1269 P 2.1 7.5 2.8 1422073_a_at Cadherin EGF LAG seven-pass G- type receptor 2 Celsr2 503 P 349 P 1164 P 454 P 2.1 0.9 2.6 1441615_at Core-binding factor, runt domain, alpha subunit 2, translocated to, 2 homolog Cbfa2t2h 287 P 159 P 219 P 85 P 2.1 2.2 4.4 1429443_at Copine IV Cpne4 2930 P 1400 P 1988 P 975 P 2.1 14 2.5 1426165_a_at Caspase 3 Casp3 213 P 103 P 772 P 373 P 2.1 0.7 0.4 1421072_at Iroquois related homeobox 5 Irx5 1753 P 846 P 3435 P 1676 P 2.1 79 12 1416572_at Matrix metallopeptidase 14 Mmp14 143 P 93 A 631 P 284 P 2.1 0.7 1.1 1416159_at Nuclear receptor subfamily 2, group Nr2f2 310 P 195 P 657 P 276 P 2.1 1.4 0.3 F, member 2 1439015_at Glial cell line derived neurotrophic factor family receptor alpha 1 Gfra1 1066 P 566 P 767 P 333 P 2.0 5.5 1.1 1421860_at Calsyntenin 1 Clstn1 572 P 326 P 2498 P 1182 P 2.0 0.9 1.0 1434421_at Immunoglobulin superfamily containing leucine-rich repeat 2 Islr2 7151 P 3144 P 11406 P 5981 P 2.0 1.1 2.3 1421594_a_at Synaptotagmin-like 2 Sytl2 1219 P 603 P 673 P 328 P 2.0 24 5.8 1418881_at EF hand calcium binding protein 2 Efcbp2 9409 P 5229 P 13343 P 5974 P 2.0 40 8.7 1422068_at POU domain, class 3, transcription factor 1 Pou3f1 1856 P 739 P 2346 P 1336 P 2.0 0.9 14 1419406_a_at B-cell CLL/lymphoma 11A (zinc finger protein) Bcl11a 860 P 525 P 1122 P 457 P 2.0 0.2 0.5 1421152_a_at Guanine nucleotide binding protein, alpha o Gnao1 4197 P 2287 P 9525 P 4632 P 2.0 1.4 0.9 1419420_at ST6 (alpha-N-acetyl-neuraminyl-2,3- beta-galactosyl-1,3)-N- acetylgalactosaminide alpha-2,6- sialyltransferase 5 St6galnac5 4369 P 2139 P 2507 P 1349 P 2.0 15 2.0

Supplementary Table 3. Transcripts decreased in the habenula of Brn3a-/- embryos. Microarray analysis was performed in duplicate on Affymetrix 430v2 arrays. Control embryos were Brn3atLacZ/+ and experimental embryos were Brn3atLacZ/-. Expression values were globally scaled to 500. For inclusion in Table S1 or S2, assayed transcripts met the following criteria: 1) Present “P” call in both samples of at least one genotype. 2) Mean expression of at least 250 in at least one genotype 3) Concordant decreased “D” call (Table S1) or increased “I” call (Table S2) in both comparisions between matched heterozygote and knockout samples. In the cases where multiple probe sets queried a single transcript, a single representative probe set was chosen based on the highest expression level. All probe sets meeting these criteria and exhibiting >2-fold change are displayed. Gene IDs are from NCBI mouse genome build 36.

*Included due to biological interest although values did not meet inclusion criterion of concordant P call in both WT samples.

Expression value, A/P call Mean ratio HT/ HB/ HB/ Probe set Gene Title Gene Symbol Het1 KO1 Het2 KO2 KO CTX Thal 1450427_at Cholinergic receptor, nicotinic, alpha polypeptide 6 Chrna6 48 A 188 P 57 A 226 P 4.0 4.5 1.3 1433578_at Solute carrier family 10 (sodium/bile acid cotransporter family), member 4 Slc10a4 276 P 678 P 344 P 1149 P 2.9 12.4 0.3 1455358_at Ataxin 2 binding protein 1 A2bp1 1199 P 3059 P 681 P 2016 P 2.7 0.2 0.2 1449571_at Thyrotropin releasing hormone receptor Trhr 171 P 524 P 100 P 189 P 2.6 3.0 3.9 1441087_at RIKEN cDNA 2810011L19 gene 2810011L19Rik 243 P 802 P 236 P 447 P 2.6 0.9 0.3 1460408_at Gamma-aminobutyric acid (GABA-A) receptor, subunit gamma 1 Gabrg1 831 P 2181 P 401 P 966 P 2.6 21.7 1.4 1423062_at Insulin-like growth factor binding protein 3 Igfbp3 151 P 267 P 52 A 248 P 2.5 0.1 0.5 1434098_at Glycine receptor, alpha 2 subunit Glra2 430 P 1233 P 339 P 704 P 2.5 0.3 0.1 1456307_s_at Adenylate cyclase 7 Adcy7 180 P 436 P 61 P 167 P 2.5 4.1 0.7 1452065_at cDNA sequence BC027127 BC027127 680 P 1885 P 522 P 1120 P 2.5 2.9 0.7 1429348_at Semaphorin 3C Sema3c 602 P 1333 P 446 P 1159 P 2.4 0.2 1.1 1416286_at Regulator of G-protein signaling 4 Rgs4 312 P 770 P 375 P 863 P 2.4 0.6 0.5 1444736_at Cadherin 7, type 2 Cdh7 149 P 364 P 69 P 151 P 2.4 5.9 1.1 1420667_at Double C2, beta Doc2b 726 P 1978 P 500 P 908 P 2.4 4.2 1.5 1449588_at ATP-binding cassette, sub-family A (ABC1), member 4 Abca4 265 P 354 P 97 A 488 P 2.3 6.0 2.7 1429887_at RIKEN cDNA 2310005C01 gene 2310005C01Rik 960 P 2091 P 665 P 1646 P 2.3 0.5 2.9 1426114_at Heterogeneous nuclear ribonucleoprotein A/B Hnrpab 1105 P 1990 P 470 P 1623 P 2.3 0.8 0.3 1456512_at PDZ domain containing RING finger 4 Pdzrn4 141 P 476 P 196 P 295 P 2.3 0.9 0.1 1455498_at G-protein-coupled receptor 50 Gpr50 129 P 293 P 94 P 207 P 2.2 13.3 3.4 1421754_at cDNA sequence AY036118 AY036118 1576 P 3155 P 723 P 1880 P 2.2 1.4 1.5 1438483_at Nitric oxide synthase 1, neuronal Nos1 266 P 474 P 283 P 707 P 2.1 0.5 2.3 1433707_at Gamma-aminobutyric acid (GABA-A) receptor, subunit alpha 4 Gabra4 196 P 535 P 176 P 260 P 2.1 0.9 0.1 1459850_x_at Glycine receptor, beta subunit Glrb 476 P 1024 P 386 M 807 P 2.1 0.7 0.6 1456398_at Taurine upregulated gene 1 Tug1 1663 P 2338 P 489 P 2228 P 2.1 2.7 2.4 1455346_at Mannan-binding lectin serine peptidase 1 Masp1 374 P 755 P 241 P 542 P 2.1 0.3 0.8 1437156_at EF hand calcium binding protein 1 Efcbp1 194 P 479 P 130 P 198 P 2.1 0.6 0.5 1447825_x_at protocadherin 8 Pcdh8 2372 P 4439 P 1574 P 3505 P 2.0 0.4 0.5 1426978_at kelch-like 2, Mayven (Drosophila) Klhl2 662 P 1404 P 361 P 646 P 2.0 0.5 0.4 1435307_at RIKEN cDNA 6430502M16 gene 6430502M16Rik 562 P 964 P 219 P 585 P 2.0 45.1 0.6 1428167_a_at myelin protein zero-like 1 Mpzl1 885 P 1689 P 543 P 1125 P 2.0 1.0 0.5 1455272_at Glutamate receptor, metabotropic 5 Grm5 1191 P 2290 P 806 P 1627 P 2.0 2.1 0.4 1444615_x_at Runt-related transcription factor 1; translocated to, 1 Runx1t1 1181 P 2228 P 543 P 1135 P 2.0 0.7 0.2

Supplementary Table 4. Transcripts increased in the habenula of Brn3a-/- embryos. Experimental details appear in Table Supplementary Table 1.